Protein crystallization

Mirjam Leunissen

An essay on several aspects of protein crystallization research

Lysozyme

Mirjam Leunissen Department of solid state chemistry October 2001 Supervisor: Willem van Enckevort

Front page: collage of protein crystals

Abstract
This essay is about the crystallization of proteins. Since the first published observation of crystallizing proteins about 160 years ago, protein crystal growth has developed into an extensive research field with many applications, for instance in the pharmaceutical industry. In this report, an overview of the unique physical-chemical characteristics of protein molecules, their properties in solution and their properties in the crystalline state, together with a description of some important aspects of protein crystallization and research in this field, is given. Seven proteins that are the main models of crystallization studies are presented, just as the general (thermodynamic) principles applying to protein crystal growth and the main parameters influencing the process. The most commonly used protein crystallization techniques are described, together with their underlying principles and the approaches used to find suitable conditions for crystallization of a particular protein. Focusing on the fundamental studies of protein crystallization on a molecular level, the current knowledge of nucleation processes, crystal growth mechanisms and kinetics is summarized. Concluding this essay, an inventory is made of knowledge that is still missing, but that is crucial in order to develop a more directed crystallization approach. Suggestions for future research, aimed at obtaining this insight, are given.

Contents
Abstract Chapter 1 Introduction
1 2 4

1.1 The importance of protein crystal growth 1.2 A history of protein crystallization 1.3 Aspects of protein crystallization research

Chapter 2

Features and properties of proteins

6 7 8 9 9

2.1 Protein structure 2.2 Properties of proteins in solution 2.3 Properties of protein crystals 2.4 The complexity of protein crystallization 2.5 Protein model systems

Chapter 3

Principles of protein crystallization

13 15

3.1 The thermodynamics of crystal growth 3.2 Parameters influencing protein crystal growth

Chapter 4

Methods and approaches in protein crystallization

18 25

4.1 Protein crystallization methods 4.2 Approaches to find crystallization conditions

Chapter 5

Nucleation and growth processes of protein crystals

28 28

5.1 Methods of investigation 5.2 Formation of critical nuclei

5.3 Growth mechanisms, the growth unit, transport processes and kinetics 30

Chapter 6 References

General discussion and conclusion

36

Chapter 1 Introduction
This essay deals with the crystallization of the members of one particular, but very broad, class of biological macromolecules: the proteins. Protein crystallization forms a very extensive field of research, with many different aspects and applications. As you will learn in this essay, it is in some respects rather different from conventional crystallization of inorganic, small molecule compounds, for instance where the crystallization techniques are concerned, while in other respects they have many characteristics in common. An example of such similarity is formed by the crystal growth mechanisms. In order to get a bit acquainted with the subject, in this introductory chapter, I will first tell something about the importance and applications of protein crystallization and the development of the field of protein crystal growth from its starting point until now. Furthermore, focusing on the research component of the protein crystal growth field, I will indicate its many different aspects and which of them will be treated in this essay.

1.1

The importance of protein crystal growth

So, question is: why would anybody want to spent (sometimes huge) efforts on the crystallization of proteins? While, in the past, purification of mixtures was the main goal in crystallizing experiments, at present the main answer to the question stated above is provided by the fact that studies of the atomic structure of biological macromolecules (i.e. proteins, DNA, RNA, etc.) have proved of great value in revealing structure/function relationships that are of importance in our understanding of how enzymes, nucleic acids and other macromolecules operate in biological systems. Recently, nuclear magnetic resonance (NMR) studies of protein solutions have yielded atomic structure information for some small proteins. However, for proteins with molecular weights in excess of 20,000 Da (Da=dalton=1.66x10-27 kg, this unit of mass is specifically applied to proteins) only X-ray and neutron diffraction techniques can provide structure information to atomic resolution. Such studies require single crystals of high structural perfection and with minimum dimension of several hundred micron. But, one may ask, is the amount of information obtained really worth the effort, time and money crystallization costs? Well, I would say yes, because crystallographic studies of biological macromolecules have become of considerable interest to pharmaceutical, biotechnological and chemical industries, as promising tools in protein engineering, drug design and other applications to biological systems (figure 1.1).

Fig. 1.1 Use of protein crystallography. Diagram showing the path by which X-ray crystallography, in combination with computer graphics analysis and synthetic chemistry, may be rationally applied to create new chemical compounds such as medicinal drugs and pharmaceuticals, pesticides and herbicides and industrial fine chemicals. Source: [1].

The elaborate information that can be obtained from the three-dimensional structure of a protein is useful in a variety of ways. From the basic biological view point, this information underlies our understanding of the mechanisms by which enzymes, receptors, hormones, etc. function in biological systems. Within the pharmaceutical industry, protein structure information can be helpful in the development of novel drugs. Since many pharmaceutical agents act by interacting with proteins, knowledge of the three-dimensional structure of a target protein can be used to design compounds that selectively bind to sites of this protein and thereby inhibit its activities. Another highly promising application of protein crystallography is in protein engineering. Using molecular biology techniques, investigators can specifically alter protein molecules by site-directed mutagenesis (i.e. by altering proteins in specific, selected regions by altering the DNA stretch encoding that part of the protein). In general, the most promising approaches to protein engineering depend upon detailed structural information about the proteins of interest. An additional use of protein crystallography is in the design of synthetic vaccines. Several recent studies have indicated that effective vaccines might be made from synthetic peptides (very small proteins) that are representative of protein segments found on the surfaces of target proteins. Protein crystallography provides one of the most effective techniques for locating those peptides. However, the crystallization of proteins is not only an excellent tool to obtain information about the three-dimensional protein structure, but also a very interesting subject for crystal growth studies in its own right. Compared to conventional, small molecule (often salts) crystal growth systems, proteins display relatively slow growth kinetics and have growth units with a large size (because individual protein molecules are very large). These facts allow one to more readily study crystal growth mechanisms, par example by means of in situ atomic force microscopy observations. Therefore, protein crystallization now serves as the best model system for general crystallization from solution. Concluding, one can say that there are two interests: a general interest in delineating processes that play a role in crystallization from solutions and an interest in the crystallization of certain proteins in particular, in order to obtain structural information about these proteins. These two interests do not stand on their own, however. As many proteins tend to resist their easy crystallization, more insight in the processes at work during crystal formation might enable a more directed approach with a higher chance of success. I will come back to this in the concluding chapter of this essay.

1.2

A history of protein crystallization

This section is based on information in [1] and references therein. In table 1.1 (page 3) a chronology of protein crystal growth is given. The first protein crystals The history of (recorded) protein crystal growth started about 160 years ago. The first published observation of the crystallization of a protein appears to be by Hnefeld in 1840. The protein, hemoglobin from the earthworm, was obtained as flat plate-like crystals when the worms blood was pressed between two slides of glass and allowed to dry very slowly. This observation clearly stated that protein crystals can be produced by the controlled evaporation of a concentrated protein solution, that is, protein crystals can be produced by slow dehydration. This is the basis for most of the techniques we use today and which are described in section 4.1. The first investigators that took up protein crystallization, focussed on hemoglobin (of many different animal species) for the next 15 years. Unfortunately, through 1850, all of the blood crystals reported, appeared to have grown more or less fortuitously and no investigator had suggested any general procedure for their directed growth. The first person to actually devise 2

successful and reproducible methods for the growth of hemoglobin crystals was Fnke, who published a series of articles on the purposeful growth of hemoglobin crystals. Following hemoglobin, the next class of proteins to be investigated, in the period from 1850 to about 1900, were the plant seed reserve proteins, principally the so called globulins. The methods that were developed to crystallize these proteins included: extraction of proteins into salt solutions followed by slow cooling, dialysis of a salt solution extract of the seeds exhaustively against distilled water and treatment of protein solutions with alcohol, acetone or ether. In these procedures we find for the first time the exploitation of several approaches now in common use: temperature variation under constant solution conditions, dialysis against low ionic strength solutions (to take advantage of the low solubility of many proteins at low ambient salt concentrations) and the use of organic solvents as precipitating or crystallizing agents. At almost the same time the work with plant seed proteins was carried out, similar efforts were done to crystallize the proteins hen egg albumin and horse serum albumin. The procedures for their crystallization used many of the suggestions of Hofmeister regarding the salting out of proteins by high concentrations of salt ions and the precipitation of proteins by careful regulation of pH (for an explanation, see section 3.2). The first enzyme, urease, was crystallized in 1925 by Sumner and at almost the same time the first hormone, insulin, was crystallized too. Crucial to the insulin crystallization was the addition of divalent zinc ions. This was one of the first examples of crystal growth promoted by the addition of metal ions.

Table 1.1 A chronology of protein crystal growth. Source: [1].

A change of attitude Until the 1930s (but also beyond that for many years), the rationale for crystallizing proteins, particularly enzymes, was to supply a technique for purifying a specific protein from a complex extract, or to demonstrate the purity of a preparation. In the late 1930s, however, certain X-ray diffractionists began to turn their attention to protein crystals as a source of structural information about biological macromolecules for reasons stated in the previous section. This interest carries on till present time. The interest of X-ray diffractionists was influential in promoting efforts to reproducibly grow high quality protein crystals, but also led to efforts to increase success rates and to automate the crystallization process. The background for the latter is formed by the fact that with the extraordinary advances in data collection and computing techniques and with the revolution in pharmacology and biotechnology, the ask for new macromolecular crystals very soon greatly surpassed their supply. At present the bottleneck in solving the problem of limited crystallization yields is mainly formed by a lack of insight. As you will see in this essay, most protein crystallization approaches are based on the trial-and-error principle, while insight in the fundamental processes at work is very restricted. As a consequence, many proteins still resist crystallization for unknown reasons. Clearly, at present, the challenge is to obtain enough knowledge about the processes at work and, based on this knowledge, to develop new, more directed methods in order to be able to readily crystallize any protein at will.

1.3

Aspects of protein crystallization research

There are many areas of research related to protein crystallization, each focusing on different aspects. In this essay I will treat only a limited number of them. Chapter 2 provides an introduction to the subject common to all of the studies: protein molecules. The general physical-chemical characteristics of protein molecules, their properties in solution and their properties in the crystalline state are dealt with. From this information, the answer to the question why the crystallization of proteins is very complex will be extracted. Furthermore, in this chapter, the seven proteins that are the main models of crystallization studies are presented. In chapter 3, I will treat the general (thermodynamic) principles applying to (protein) crystallization and explain which parameters play an important role in the crystallization processes and how they exert their influences. Chapter 4 is an overview of modern protein crystallization techniques, their respective advantages and/or disadvantages and the underlying thermodynamic principles. Moreover, in this chapter, the approaches used at present to find suitable conditions for the crystallization of a particular protein are sketched. Then, in chapter 5, I will summarize the current knowledge of nucleation processes, crystal growth mechanisms and kinetics of proteins and how they can be studied experimentally. To conclude this essay, I will reflect on all previous chapters, in the general discussion and conclusion of chapter 6. This, in order to make an inventory of crucial knowledge that is still missing for a more directed approach. I will also sketch what, in my opinion, the direction of future research should be. Of course, there are many topics left which I will not treat, like: polymorphy, defects in protein crystals, sources of impurities, purification of protein samples, automation of crystallization trials and mutagenesis of proteins in order to transform it into a protein more suitable for crystallization. Also, the discussion presented here will not consider the possibility of conformational heterogeneity and flexibility of the protein molecular structure, which may be important for the perfection and utility of certain protein crystals. However, below, I will give references that can guide you to literature about some of these topics. A comment (short or more detailed) on virtually each of all aforementioned topics, together with lots of references to relevant literature can be found in a book written by McPherson [1]. Especially 4

if you are new to the field of protein crystallization I would highly recommend it. Other interesting references are: [2] and [3], if you are interested in protein molecular interactions in solutions and [4] for interactions in the crystalline state. For a general theoretical introduction to the problem of protein nucleation, see [4], [5], [6], [7], [8]. A comprehensive list of references on impurity effects and defect formation is provided in [9], but lots of information on these topics can also be found in [10] and [11]. A nice review of theoretical models that are used to study protein crystal formation and the major ideas used in their development is presented in [5]. Undoubtedly, the information in this essay and in the references given above will not cover the whole field of protein crystallization, but it will provide a good starting point for anybody interested in this extensive field.

Chapter 2 Features and properties of proteins

2.1 Protein structure

Proteins are so called macromolecules, because the particle diameter is ~30-100 as compared to ~3 for most inorganic particles. Proteins can be considered as polymers of amino acids, linked together in a chain-like arrangement. The number of amino acids constituting one protein molecule ranges approximately from 100 to 27,000 for the proteins known. The specific sequence of amino acids in a protein is called its primary structure.
a
+

R1 H3N C H COO+
+

R2 H3N C H COO-

R1 O
+

R2 N C H H COO+ H2O

H3N C H

Natural proteins are built up by multiple numbers of twenty different amino acids, sometimes referred to as residues. The general structure of an amino acid can be seen in figure 2.1a. In each amino acid, the amino and carboxyl groups make one bond each with the so called -carbon atom, C. One of the other two bonds of C is occupied with hydrogen, while the other is occupied with a relatively small group, called the side chain, which is different for each of the twenty amino acids. These side chains consist of simple hydrocarbon groups, that may contain aromatic rings, nitrogen, oxygen and sulfur (figure 2.2). The so called peptide bonds between the amino and carboxyl groups of neighboring amino acids (figure 2.1a) form the backbone along the polypeptide chain. The side chains form its short branches (figure 2.1b).

Ri H

(NH CH C)n OH

Fig. 2.1 Primary structure of proteins. a) Formation of a peptide bond between two amino acids that differ in their side chains (R1, R2). The carbon atom in the middle of each amino acid structure is called C. b) Overall formula for a polypeptide chain (backbone) with side chains Ri. Adapted from: [12].

The same amino acid sequence (that is, the same primary structure) can occur in different conformations through different azimuthal rotations of the side chains about peptide links. This can determine some secondary structure of the backbone, such as the formation of helical windings and sheet-like structures. In the latter case, the polypeptide chain is folded several times back and forth in a certain plane. For examples of these common types of secondary structure I would like to refer to figures 2.9a and 2.7 (page 11). Very important to the overall structure of a protein, is the fact that the amino acid side chains can both consist of polar functional groups or nonpolar functional groups (review
Fig. 2.2 Natural amino acid side chains. Chemical structure of the side chains of the 20 amino acids that form proteins. Charged (polar) functional groups in bold. Source: [12].

figure 2.2). In their natural aqueous environment, the polar groups prefer to associate with water. In contrast to these hydrophilic groups, the nonpolar hydrophobic groups prefer to associate with themselves. As a result of these preferences the molecule lowers its free energy by folding to a specific three-dimensional structure, the so called tertiary structure. The pictures in section 2.5 provide many examples of this overall three-dimensional structure of proteins. Molecules with comparable numbers of polar and nonpolar residues fold into globular shapes in which the hydrophilic residues tend to concentrate on the surface and the hydrophobic groups in the core. This three-dimensional structure is stabilized by various close-range interactions between atoms that are distant on the backbone. These include electrostatic interactions between charges and dipoles, van der Waals forces, hydrogen bonds, hydrophobic interactions and (covalent) disulfide bonds. The three-dimensional structure described above represents the final folding of a polypeptide chain, but some proteins display another level of organization, the so called quaternary structure. This refers to the fact that many functional proteins are complexes of smaller subunits. Each subunit is a polypeptide chain with its own specific tertiary, three-dimensional structure. To form the final functional protein a number of subunits associate to one big complex by means of noncovalent interactions. This arrangement of subunits is called the quaternary structure of a protein. A very nice example of the quaternary structure of a protein is displayed in figure 2.9 (page 11).

2.2

Properties of proteins in solution

The properties of a protein are to a great extent determined by the amino acid side chains present on its surface. Within the group of polar residues there are two classes of charged amino acids: acidic and basic. At neutral pH, which centers the normal physiological range, the acidic amino acids are negatively charged and the basic amino acids carry a positive charge. Therefore, at pH ~6-7 and temperatures 0-40C, typical of biological conditions, the molecular surface usually is charged. Like all polar side chains, the charged groups interact extensively with water and tend to solvate the protein. Moreover, exposed hydrophilic groups on macromolecules can bind, both transiently and in a stable manner, not only water molecules but also a variety of ions, both cations and anions. Thus, as a consequence of their polyionic character, the surfaces of macromolecules display a variegated pattern of positive and negative charge. As an example, the charge distribution on the surface of an arbitrary protein is visualized in figure 2.3. The actual net surface charge is dependent on the amino acid sequence and environmental factors, such as the pH of the solution. Lysozyme, for instance, has 10-12 positive charges at pH 4.5 [13]. As a consequence, under these solution conditions repulsion dominates the interactions between these macro-ions.

Fig. 2.3 Protein surface charge distribution. Positively and negatively charged amino acid side chains on a protein give rise to a characteristic electrostatic surface like that shown here for the protease from P. cyclopium. Blue represents positive field strength and red negative. The surface, however, can change as a function of the pH, which causes ionizable groups to gain or lose protons. Source: [1].

At the iso-electric point, pI, (at pH=11.3 for lysozyme [14], between 5 and 7 for most other proteins) there are equal numbers of positively and negatively charged residues and as a consequence at this pH value the net surface charge vanishes. However, one must realize that this does not mean that individual surface patches do not carry a charge. The net surface charge of a protein molecule changes with the pH of the solution and consequently, as one can imagine, the solubility of the protein changes with the pH too. For a particular protein, with its own specific sequence of amino acid side chains, the solubility dependence on the solution pH is given by the following equation [15]:

[ P ] = [ P ] + [ Pi ] + [ Pj ] = K [1 +
i j

( Ki /[ H + ]i) + ([ H + ] / Kjj )] exp( 0 s 0 / kT )

[P] = the total concentration of dissolved molecules [P] = the zwitterionic state of the protein molecule [Pi] = negatively charged states of the protein molecule [Pj] = positively charged states of the protein molecule

0 = the surface free energy of a protein molecule in solution s0 = the surface area of a protein molecule in solution = activity coefficient Kx = (de)protonation reaction constant side chain

According to this formula, solubility reaches a minimum at proton concentrations corresponding to the isoelectric point and steeply increases at both lower and higher pH. The temperature dependence of solubility is determined by the temperature dependence of the protonation and deprotonation reaction constants of the side chains. For proteins, which exist predominantly in an aqueous environment, one free energy minimum is represented when they are fully solvated, but in extremely concentrated solutions where there is insufficient water to maintain hydration, the molecules may aggregate as an amorphous precipitate or they may crystallize. Let us therefore consider some properties of protein crystals.

2.3

Properties of protein crystals

Protein crystals differ from crystals of most other compounds in several respects [1], [16]. One of the major differences is the high solvent content of protein crystals, which is responsible for most of their unique features. On crystallization, proteins form a wide open network of macromolecules (figure 2.4). Depending on the specific protein, between 25 and 85 vol% of the crystal consists of channels. These are lined with protein-bound water, referred to as ordered or bound water [19], and possibly some precipitate ions. This bound water forms the ordered hydration shell of the protein and is a real component of the macromolecules structure. Besides channels, also cavities are being formed and both are filled with so called disordered bulk water. Furthermore, water molecules of ill-defined character are present that provide continuity between the bulk and bound water. Much of the ordered water, localized in areas between adjacent protein molecules in the crystal lattice, plays a role in the formation of extended patterns of hydrogen bonds that supplement and strengthen the direct interactions which occur between the protein molecules [20].
Fig. 2.4 Structure of protein crystals. Schematic representation of protein crystal structure with wide open network of protein molecules lined by layers of bound water, and channels formed by the protein-bound water network filled with solution (bulk water). Source: [18]. Inset: view of monomer packing in the unit cell of a crystal of chymotrypsinogen, showing the large solvent channels penetrating the unit cell. Source: [17].

Protein-protein contacts in crystals are complex, involving a delicate balance of specific and nonspecific, intrinsically flexible interactions [5], [20]. Hydrogen bonding and electrostatic interactions, involving the participation of flexible amino acid side-chains on the proteins surface, together with numerous solvent molecules or ions that are immobilized between molecules during crystal lattice formation, are examples of specific interactions. Nonspecific interactions are van der Waals interactions and hydrophobic interactions. It is not clear from the literature whether any particular type of interaction makes a dominant contribution to the energy of protein-protein contacts in crystals. It appears that interactions between proteins represent a delicate balance of many contributions whose relative importance varies from case to case.

2.4

The complexity of protein crystallization

In general, the crystallization of proteins is a very complex process. Experiences of many investigators point out that most proteins are difficult to crystallize and even if a protein tends to crystallize relatively easily there are many parameters that must be taken into account. From the preceding sections one can identify multiple reasons for the difficulty of protein crystal growth. Apparently, protein molecules are very complex (large, flexible molecules often composed of several subunits), relatively chemically and physically instable (unfolding, hydration requirements, temperature sensitivity) and they have dynamic properties. If the solution changes, the molecule properties (e.g. conformation, charge and size) change too. Furthermore, every macromolecule is unique in its physical and chemical properties because every amino acid sequence produces a unique three-dimensional structure having distinctive surface characteristics. Thus, lessons learned by investigation of one protein only marginally apply to others. For a crystal to form, interactions between protein molecules must be suitable in their geometric arrangement, degree of specificity and strength. The size and complexity of proteins is reflected in their ability to form many different intermolecular contacts, yet only a few of these contacts must exist in a given crystal and consequently the correct selection must be made each time a molecule is incorporated [4]. Moreover, the ability to form the right interactions is not a property of the protein molecule alone, but a joint property of the protein and its solution environment. The diversity of chemical groups involved in the contacts implies that slight changes in pH, ionic strength, temperature or concentration of an auxiliary ion or molecule may strongly influence crystallization. It is not yet possible to predict the conditions required to crystallize a protein from its other physical properties. Changes in a single experimental parameter can simultaneously influence several aspects of a crystallization experiment. For example, temperature changes affect protein solubility, rates of nucleation and growth, and equilibration of the experimental apparatus. The interaction of parameters makes it difficult to design experiments to isolate individual effects and likewise complicates the interpretation of experimental results. Therefore, our insight in protein crystallization processes is still very limited and, consequently, protein crystal growth remains more of an art than a science.

2.5

Protein model systems

As can be seen in the historical table of section 1.2 many different proteins have been crystallized. Nowadays, databases contain structures of more than 10,000 different proteins, determined by Xray diffraction analysis of their crystals. However, for the study of the processes at work in protein crystal growth, only a couple of proteins are used as model systems. These proteins are relatively easy to crystallize and are thought to be representative of protein molecules in general. In the following, I will give a (short) description of the most commonly used model proteins. Each of these is accompanied by a list of references that contain further information. The space groups in which they crystallize are enlisted in table 2.1 (next page), together with the respective unit cell 9

dimensions and angles. Information on other proteins and their crystal structures can be found in the Protein Data Bank [21]. This is also the source of all pictures in this section.

Table 2.1 Crystal structures of frequently used model proteins. Only angles 90 are denoted.

Protein
Lysozyme

Space group
P1 P21 P212121 P6122 P43212

a []
27.28 28.00 59.40 87.01 79.10 126.35 105.99 136.80 136.50 88.70 61.36 71.29 118.70 78.80 44.30 117.70 58.60 182.90 147.23 87.80 173 203.60 111.84 49.24 82.5 61.23

b []
31.98 62.29 68.70 87.01 79.10 126.35 105.99 136.80 150.30 86.50 85.46 71.65 101.38 79.30 63.70 44.90 58.60 182.90 147.23 140.60 173 203.60 111.84 60.94 82.5 61.65

c []
34.29 60.50 30.80 70.4 37.90 51.64 105.99 75.70 133.40 62.50 91.46 190.96 111.97 133.30 72.70 38.00 151.80 182.90 152.58 232.40 273 144.60 249.91 48.18 34.00 48.05

[deg]
88.5

[deg]
108.6 90.8

[deg]
111.8

120 120 120

Canavalin

P63 P213 R3 C2221 I222 P21212 P212121 C2221 P1 P212121 C2 P41212

Concanavalin A

97.10

90.20 94.00

97.50

Thaumatin

(Apo-)ferritin Catalase

F432 P4212 P212121 P3121 I41 P6222

120 120 95.80 110.50

Insulin

P21 R3 P1211

Fig. 2.5 Lysozyme. This ribbon model displays the folded structure of hen egg white lysozyme. Some secondary structure in the form of helices is clearly visible.

Lysozyme The protein first studied by X-ray diffraction and still most widely used for crystal growth studies is hen egg white (HEW) lysozyme. This is an enzyme which hydrolyzes polysaccharides in bacterial cell walls. HEW lysozyme is composed of a single chain of 129 amino acids and has a molecular weight, MR, of 14,296 Da (Da=dalton= 1.66x10-27 kg). Figure 2.5 illustrates its three-dimensional, folded structure as a ribbon model of its peptide backbone. Lysozyme is particularly attractive for crystal growth research, because detailed information exists on its thermophysical properties (contrary to the situation for all other proteins). References: the crystallization of lysozyme was first published by Abraham and Robinson in [22]. Information on the solubility of the protein as a function of ambient salt concentration, pH and temperature can be found in [23], [24], [25], [26], [27], [28], [29], [30], [31].

10

Canavalin Canavalin is the major reserve protein of the seeds of the jack bean. It is composed of three identical subunits, each with MR ~47,000 Da, arranged about a perfect threefold axis. Each of the subunits consists of two chains, called A and B (figure 2.6). References: the crystallization of canavalin was first published by Sumner and Howell [32]. More information about its crystallization can be found in [33] and [34]. Studies of the solubility of canavalin are [23] and [35].

Fig. 2.6 Canavalin. a) Ribbon model displaying the rather complex quaternary structure of canavalin. The protein is a complex of three subunits, densely packed together. b) Each of the subunits is composed of two chains, called A and B.

Concanavalin A Concanavalin A is a derivative of the enzyme chitinase and is, just as canavalin, obtained from the jack bean. The protein consists of two chains (A and B) of 237 amino acids each ( and MR ~25550 Da). Figure 2.7 is a ribbon model of this protein. References: the crystallization of concanavalin A was first published by Sumner [36]. A later investigation of the proteins structure can be found in [37]. Solubility information was published by Mikol and Gieg [38].
Fig. 2.7 Concanavalin A. Ribbon model displaying the folded structure of the complex of two chains that form concanavalin A. Pronounced secondary structure in the form of sheets (indicated with arrows) is visible. Fig. 2.8 Thaumatin. Ribbon model. This relatively simple protein consists of a single chain only.

Thaumatin Thaumatin (figure 2.8) is a 207 residues, 22,203 Da, sweet-tasting protein from the African serendipity berry and has commercial potential as a sweetener. It is problematic to crystallize under most conditions, except when tartrate is added to the solution, then it will readily crystallize. References: an X-ray analysis of thaumatin has been published in [39]. Phase diagrams of the protein can be found in [40]. a Ferritin Ferritin is the iron storage protein of higher organisms. It binds and thereby stores iron intracellularly in the liver. Apoferritin is the same protein but with the iron ion (present in the structure of ferritin) removed. This protein is a very large oligomer composed of 24 identical, 174 residue subunits arranged in an octahedral complex, resulting in a total molecular weight of ~450,000 Da. An image of this impressive protein can be seen in figure 2.9. Ferritin is among the most readily crystallized of all proteins, because it crystallizes almost immediately when Cd2+ ions are added to a solution of this protein. References: the crystallization of ferritin was first published by Laufberger [41]. Physical and chemical properties of the protein can be found in [42].
Fig. 2.9 Ferritin. a) Ribbon model of a single 174 residue subunit of the ferritin protein. Secondary structure almost entirely consists of helical windings. b) Ferritin, composed of 24 of the subunits displayed in (a). The helices of the subunits are depicted as cylinders. Note the globular shape of the oligomer.

11

Insulin Insulin is a hormone from the pancreas. It consists of two chains (A and B) of respectively 21 and 30 amino acids (figure 2.10a). These chains are linked together by two (covalent) disulfide bonds. The proteins molecular weight is ~ 7,300 Da. Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the B chains. One such dimer is depicted in figure 2.10b. In the presence of zinc ions insulin may even form hexamers, as displayed in figure 2.10c. References: the crystallization of insulin was first published by Abel et al. in [43]. Further investigations on crystalline insulin are presented in [44] and [45].

a)

b)

c)

Fig. 2.10 Insulin. Ribbon model. The insulin monomer consists of two chains (A and B) covalently linked together via so called disulfide bonds. In aqueous environment insulin tends to form dimers due to hydrogen bonding between the B chains of the monomers. In the presence of divalent zinc ions hexamers are formed. The zinc ions are visible in the structure as grayish balls.

Catalase Catalase is a detoxifying, heme-containing enzyme from the liver. It is responsible for the elimination of hydrogen peroxide without formation of free radicals. Catalase consists of 4 identical subunits of 484 residues and 55472 Da each (figure 2.11). References: the crystallization of catalase was first published by Sumner and Dounce [46].

b
Fig. 2.11 Catalase. Ribbon model. a) A single 484 residue subunit of the catalase protein. b) The complex quaternary structure of catalase. The protein consists of a packing of 4 of the subunits displayed in (a).

12

Chapter 3 Principles of protein crystallization

In this chapter, I will introduce the general (thermodynamic) crystal growth principles and parameters influencing crystallization processes. The crystallization methods described in the next chapter are based on these principles and parameters. For a description of the specific nucleation processes, crystal growth mechanisms and kinetics of proteins I would like to refer to chapter 5.

3.1

The thermodynamics of crystal growth

Crystallization is a complex process, involving multiple equilibria between different states of the crystallizing species [6], [47], [48]. The three stages of crystallization common to all molecules are nucleation, crystal growth and cessation of growth. During nucleation enough molecules associate in three dimensions to form a thermodynamically stable aggregate, the so called critical nucleus. These nuclei provide surfaces suitable for crystal growth, which can occur by a couple of different mechanisms. Crystal growth ceases when the solution is sufficiently depleted of protein molecules, deformation-induced strain destabilizes the lattice, or the growing crystal faces become poisoned by impurities. Both crystal nucleation and growth occur in supersaturated solutions where the concentration of the crystallizing species, in our case protein, exceeds its equilibrium solubility value. The region of solution parameter space suitable for crystallization is generally represented on the phase diagram by the solubility curve (figure 3.1). The supersaturation requirements for nucleation and crystal growth differ. This is shown on the phase diagram where the supersaturation region is further divided into regions of higher supersaturation (the labile region), where both growth and nucleation occur, and lower supersaturation (the metastable region), where only growth is supported.

Supersaturation

Supersolubility curve

Labile region -stable nuclei spontaneously form and grow

Solubility curve

Metastable region -stable nuclei grow but do not initiate

Fig. 3.1 Phase diagram applying to crystal growth. The solubility curve (solid) divides phase space into regions that support crystallization processes (supersaturated solutions) from those where crystals will dissolve (unsaturated solutions). The supersolubility curve (dashed) further divides the supersaturated region into higher supersaturation conditions where nucleation and growth compete (labile phase) and lower levels where only crystal growth will occur (metastable phase). Different kinds of solubility decreasing parameters will be treated in the next section. Adapted from: [47].

Unsaturation

[Macromolecule]

Now, the strategy employed to bring about crystallization is to guide the system toward a state of reduced solubility (i.e. supersaturation) by modifying the properties of the solvent or the character of the macromolecule (protein crystals are always grown from solutions, because this is the only environment in which protein molecules are stable, that is, remain folded in their native conformation). This can be done by changing one or more of many different parameters, using one of several methods possible. This will be explained in detail in sections 3.2 and 4.1. 13

Fig. 3.2 Reversible molecular association reactions involved in the assembly of crystals. Monomers initially combine into small aggregates (here, called chains). The association of monomers into chains leads to the formation of prenuclear aggregates that continue to grow by further addition of monomers or chains. When sufficient molecules associate in three dimensions, a thermodynamically stable critical nucleus is formed. The addition of monomers and/or chains to critical nuclei eventually leads to the formation of macroscopic crystals. Source: [6].

Monomers

Prenuclear aggregate

Critical nucleus Crystal

Associated chains

The solubility must be decreased very slowly, otherwise amorphous precipitate will form instead of nicely ordered crystals (this is a major problem with macromolecular crystallization, while seldom encountered in the crystallization of conventional molecules). Moreover, care must be taken that the supersaturation level does not become too high. Indeed, a very high supersaturation level increases the chance of obtaining any crystals, but it has some possible deleterious consequences too. Too large numbers of nuclei may be produced, leading to many small crystals or the crystal growth process may become unfavorably rapid, leading to very low quality crystals. The ideal approach would be to induce nuclei at the lowest level of supersaturation which permits their formation, that is, just within the labile region. As these few nuclei begin to grow, the system will gradually enter the metastable region, due to depletion of solute from the solution. In the metastable region the few select crystals continue to develop, while no new nuclei or precipitate can occur. Indicative for the complexity of crystal growth processes is the fact that the formation of a critical nucleus may come about in many different ways. This is, because complex equilibria between many different states of a protein exist in a solution (figure 3.2). Inelastic light scattering investigations have shown that, during the course of crystallization, not only monomers, but also higher-molecular-weight aggregates and clusters of macromolecules with a broad distribution of sizes are present in solution. These may be discrete, ordered, prenuclear aggregates of subcritical size, they may be disordered clusters formed from a variety of nonspecific interactions between macromolecules, linear aggregates on a pathway toward precipitate, or only various dimers and trimers associating transiently through diverse interactions. With time, some of these aggregates may grow and ultimately give rise to crystal nuclei. Others may become ordered on the surfaces of existing crystals and contribute to their growth. Still others may form amorphous precipitates or other energetically favorable but for crystal formation competitive phases. Thus, the pathway from monomeric proteins to crystals is not necessarily a straight one, but generally involves many different processes and equilibria.

14

3.2

Parameters influencing protein crystal growth

A wide variety of physical, chemical and biochemical parameters affect protein crystallization processes, as can be seen in table 3.1. Of course the physical-chemical characteristics of the crystallizing protein, described in chapter 2, are important, just as the purity of the sample, the properties of the solution used for crystallization (e.g. composition, temperature and pH) and the method of sample handling. In the following, I will shed some light on the role of many (mainly physical and chemical) factors, given a specific protein sample (with a particular purity, sequence, modifications etc.). Some factors will also be shortly commented on in section 4.1. An extensive review of the influence of all factors, including the biochemical ones, can be found in references [48] and [50].
Table 3.1 Crystallization parameters. Physical, chemical and biochemical factors affecting the outcome of a protein crystallization experiment. Adapted from: [48] and [49].

Physical factors
Temperature Methodology Time Pressure Gravity, convection and sedimentation Vibrations and sound Magnetic fields Electric fields Dielectric properties Viscosity Equilibration rate Nucleants Volume Particulate/amorphous material Surface of crystallization device Sample handling

Chemical factors
Precipitant type Precipitant concentration pH and buffer Ionic strength Reducing/oxidizing environment Sample concentration Metal ions Detergents Small molecule impurities Polyions Crosslinkers Heavy metals Reagent source Reagent purity Reagent formulation

Biochemical factors
Sample purity Macromolecular impurities Aggregation Posttranslational modifications Sample source Sample storage Proteolysis Chemical modifications Sequence modifications Sample symmetry Sample pI Sample history Ligands, co-factors, inhibitors Microbial contamination Purification method

pH and temperature As we learned in the previous section the objective in crystallizing a protein is to gradually force the macromolecule from solution by decreasing its solubility (that is, increasing its supersaturation). Many factors can influence protein solubility, which depends on the proteins surface charge (section 2.2). A proteins solubility is usually quite sensitive to pH and to temperature as well in many cases [51], [52]. For many years it was thought that the optimal pH target for crystallization of a protein should be its isoelectric point, pI. This was intuitively appealing because at its pI, a macromolecule carries an equal number of positive and negative charges and is therefore electrostatically neutral. This would seem to be the best situation for mutually attractive electrostatic interactions. In addition most macromolecules do have a pronounced solubility minimum at their pI values and some even precipitate at their pI. Eventually, however, an analysis was conducted based on an early version of the NIST/CARB BMC Data Base, which contains information on the crystallization conditions of many thousands of proteins [53]. The outcome revealed that no correlation between the pI of crystalline proteins and the pH at which they were crystallized existed. The pI even appears inclined toward formation of amorphous precipitate over the crystalline state. Rather, most proteins were successfully crystallized near their physiological pH. Therefore, at present, the seemingly best approach is to crystallize a protein from a solution near its physiological pH. This has as advantage that the risk of denaturation (i.e. the unfolding of the protein under conditions where it is no longer stable) is minimized. 15

In section 2.2, it was explained that the temperature dependence of protein solubility is determined by the temperature dependence of the protonation and deprotonation reaction constants of the amino acid side chains in the protein structure [15]. In spite of the fact that most proteins display a clear solubility dependence on temperature, this parameter is not very often used to control supersaturation, but is in the majority of the cases kept constant during the entire experiment. Crystallization has been reported to occur over the entire range from 0 to 40C and in some cases even 60C, although it is usually conducted at either 4C or at room temperature, 25C. Just as is the case with the pH, extremes in temperature tend to cause denaturation of proteins [1]. Precipitants Protein solubility can also be decreased by changing the composition of the solution, for instance by inclusion of additives such as alcohols, hydrophilic polymers and detergents. These solubilityinfluencing agents are commonly known as precipitants [1], [54]. Protein precipitants fall into four broad categories: salts, organic solvents, (long-chain) polymers and non-volatile organic compounds. In table 3.2 the most common precipitants in each of these categories (the last two have been taken together) are enlisted.
Table 3.2 Precipitants used in macromolecular crystallization. Adapted from: [48] and [50].

Salts
Ammonium or sodium sulfate Lithium sulfate Lithium chloride Sodium or ammonium citrate Sodium or potassium phosphate Sodium or potassium or ammonium chloride Sodium or ammonium acetate Magnesium or calcium sulfate Cetyltrimethyl ammonium salts Calcium chloride Ammonium nitrate Sodium formate

Organic solvents
Ethanol Isopropanol 1,3-propanediol 2-methyl-2,4-pentanediol Dioxane Acetone Butanol Acetonitrile Dimethyl sulfoxide 2,5-hexanediol Methanol 1,3-butyrolactone Poly(ethylene)glycol 400

Polymers
Poly(ethylene)glycol 1000, 3350, 6000, 20000 Jeffamine T Polyamine

Salts exert their influence as precipitant according to the salting out principle [55]. The solubility of a protein exponentially decreases as the ionic strength of the solution is increased. The rate of decrease is a function of the particular protein and ions involved. An example of the solubility of lysozyme as a function of varying concentrations of different salts is given in figure 3.3 (page 17). A simple explanation for the salting out effect is that water molecules, otherwise available for solvation of the protein, are monopolized to form bonds with the small ions, thus the salt dehydrates the protein. When the concentration of ions becomes sufficiently high, the proteins are driven to neutralize their surface charges by interacting with one another. This interaction may result in an ordered arrangement of the proteins in a crystalline lattice. The order of salting out effectiveness of (an)ions follows the Hofmeister series [56], SO42- > HPO42- > CH3COO- > citrate3- > tartrate2- > HCO3- > CrCO3- > Cl-> NO3- > ClO3-, for negatively charged proteins, but is reversed for positively charged ones [52], [57]. Divalent and trivalent ions, such as sulfate and phosphate, are most commonly used. Cations are less effective in salting out. A theoretical discussion of the effect of ionic strength on the nucleation of protein crystals is presented in [58].

16

Fig. 3.3 Lysozyme solubility. a) Solubility of lysozyme (mg/ml) as a function of salt concentration, expressed in moles/liter (M) at 18C and pH 4.5. b) Solubility of lysozyme (mg/ml) as a function of salt concentration, expressed in moles/liter (M) at 18C and 40C and at pH 4.5. Lysozyme/NaCl data are given as an internal reference. Source: [1].

The addition of certain organic solvents may produce crystallization by a similar competition for water, due to hydrogen bonding properties of these compounds, but also by hydrophobic exclusion of protein solutes (excluded volume effects). Moreover, they significantly reduce the dielectric screening capacity of the solvent. This leads to an increase of the effective strength of the electrostatic forces between individual protein molecules, thereby enhancing their mutual attraction. The mechanisms by which (long chain) polymers induce proteins to crystallize are not fully understood. It certainly involves lowering the dielectric constant of the medium and it probably shares some characteristics both with salts that compete for water and with other precipitants that rely on volume exclusion. Through excluded volume effects proteins are dehydrated, because the extended chain polymer and its entrained water are excluded from the area near the protein. Other parameters As a possible component of crystallization solutions, various metal ions have been observed to induce or contribute to the crystallization of proteins [1], [48], [59]. In some instances these ions are essential for the proteins biological activity and it is, therefore, reasonable to expect that they might aid in maintaining certain structural features of the molecule. In other cases, however, metal ions, particularly divalent metal ions of the transition series, were found that stimulated crystal growth but played no known role in the macromolecules activity. Metal ions may bridge and stabilize intermolecular contacts in crystals. Of course the protein solution must have a concentration high enough to enable crystal nucleation. The most common range is 5 to 30 mg/ml. From prior experience it is known, that for the growth of large, single crystals, once conditions have been identified, crystallizations are best carried out in the range of 3-10 mg/ml. This usually yields the most reproducible and consistent results. The factors elaborated on above are the most well-known to influence protein crystallization. However, of many other factors it is not exactly known which influence they exert, or how they exert it. Until now, there have been virtually no systematic studies of such factors as pressure, sound, vibrations, electrical and magnetic fields on the nucleation, rate of growth or final quality of protein crystals. 17

Chapter 4 Methods and approaches in protein crystallization

4.1 Protein crystallization methods

In the previous chapter, many parameters that affect a proteins solubility and its crystallization process were described. Based on these parameters, many different approaches for creating supersaturation and thus initiating the nucleation process are possible. Some of them are mentioned in table 4.1.
Table 4.1 Approaches for creating supersaturation. Source: [1].

Directly mix macromolecule with excess precipitant to immediately create a supersaturated condition Alter temperature Increase salt concentration (salting out) Increase or decrease pH Add a ligand that changes the solubility of the macromolecule Alter the dielectric of the medium Physically remove water (evaporation) Add a polymer that produces volume exclusion Add a cross-bridging agent Concentrate the macromolecule Remove a solubilizing agent

Sometimes the same approach has been implemented in a slightly different form in several crystallization methods, leading to a rather large number of techniques. More than 20 techniques have been reported and these are listed in table 4.2, together with the number of macromolecules and crystal forms successfully crystallized by each of these methods. In figure 4.1 (page 19) comparable techniques (like bulk dialysis and microdialysis) have been grouped together. Comparison learns that batch methods, vapor diffusion and dialysis are most commonly used.
Table 4.2 Total number of crystal forms reported grown by various techniques. Data from the NIST/CARB/NASA Biological Crystallization Database (as of November 1995). Source: [49].

Crystallization method
Bulk dialysis Batch method Vapor diffusion Microdialysis Vapor diffusion on plates or slides Vapor diffusion in hanging drops Free interface diffusion Dialysis against distilled water Dialysis Microbatch Vapor diffusion in Lagerkvist cells Concentration by evaporation Temperature crystallization Dialysis against low ionic strength Electrodialysis against tap water Liquid bridge pH induced crystallization Diffusion in capillaries Direct addition of precipitant Vapor diffusion with microscopic seeding Concentration by ultracentrifuge Macroseeding Microcapillary batch method

No. of macromolecules
63 245 163 119 244 534 45 16 44 5 11 96 18 2 1 1 1 2 7 17 2 43 2

Total crystals
71 385 197 145 332 635 58 23 55 5 11 104 36 2 1 2 1 2 8 19 2 44 2

18

Fig. 4.1 Protein crystallization methods. Histogram showing the relative successful use of the various methods for macromolecular crystallization. Currently, vapor diffusion methods are far more popular than any other approach. Source: [1].

In the following, I will give a description of the most common and also some less common, but still suitable, protein crystallization methods. For each method the general principles are outlined and in most cases illustrated by means of a phase diagram. The circles on the phase diagrams indicate the levels of supersaturation that occur in the various crystallization solutions. Specific requirements, advantages and disadvantages of methods are given. The information was obtained from references [1], [4], [47], [50] and [60]. A description of techniques that are not further elaborated on here, can also be found in these publishings. For experimental protocols of the techniques I would like to refer to [61] and [62]. Temperature induced crystallization Temperature as parameter for supersaturation control is particularly attractive because heat diffuses in aqueous solutions at least two orders of magnitude more rapidly than species, it can easily be applied to closed systems and precise temperature control and programming technology is readily available. In fact, many of the earliest examples of protein crystallization were based on the creation of concentrated solutions at elevated temperatures followed by slow cooling [1]. However, in spite of its many advantages, temperature is not very often used for supersaturation control nowadays. This is very unfortunate, because it has already proved to be a particularly powerful approach for the crystallization of most other kinds of molecules. Moreover, because temperature is relatively easy to set and program precisely, it is an attractive control parameter to optimize conditions separately for nucleation and growth, for growth of good quality crystals requires lower supersaturation levels than the formation of initial nuclei (section 3.1), [63], [64]. A recent example of temperature induced crystallization can be found in [23]. Evaporation induced crystallization

Another classic procedure for inducing proteins to crystallize is to (gradually) increase the level of saturation of a salt, thereby reducing the proteins solubility (salting out, section 3.2). Traditionally the salt has been ammonium sulfate, but others are also in common use (table 3.2, page 16).
In this category, evaporation is perhaps the most primitive method to increase the salt concentration. It simply requires that the mother liquor be allowed to slowly evaporate, under nondenaturing conditions, so that the salt (and protein) concentration rises to produce supersaturation. Evaporation can be achieved simply by exposing the crystallizing solution to open air, but, as you will see later in this section, more advanced set ups are far more popular. 19

Batch crystallization Very simple too, are batch crystallization methods (figure 4.2). All components are directly combined into a single, supersaturated protein solution, which is then left undisturbed. The technique can be miniaturized by immersing protein droplets as small as 1 l into an inert oil. The oil prevents evaporation of the sample. This is the so called microbatch method [65]. Besides the very limited amounts of sample needed, the latter method has as further advantage that the samples are protected from airborne contamination, as they are never exposed to the air during the experiment.

Batch crystallization
Supersaturation

Initial conditions

Final conditions

Microbatch crystallization

Unsaturation

Initial conditions

Final conditions

[Macromolecule]

Fig. 4.2 Batch crystallization. Schematic diagram of batch and microbatch crystallization experiments. In batch experiments, vials containing supersaturated protein solutions are sealed and left undisturbed. In microbatch methods, a small (2-10 l) droplet containing both protein and precipitant is immersed in an inert oil which prevents droplet evaporation. In batch methods, the initial solution is located within the labile region of the phase diagram (solid circle). Depending on the concentration of the solution following completion of crystallization, the equilibrium solution concentration of protein is likely to have decreased so that the solution is now within the metastable region (open circle). Source: [47].

Given the static nature of the batch crystallization experiment, success requires that supersaturation levels sufficient for nucleation be achieved on mixing. This means that relatively high concentrations of the ingredients come into contact with each other upon being dispensed simultaneously. This can cause shock nucleation leading to the production of crystal showers or even precipitation of the protein. The fact that the solution remains the same during the experiment is both an advantage and a disadvantage. On the one hand, one exactly knows the conditions at which crystallization occurs, on the other hand one must be very close to the conditions which promote crystal growth in order for this technique to be successful. If one has the desire to change the conditions during the experiment it involves a disturbance of the crystallization drop itself. Vapor diffusion Contrary to batch methods, vapor diffusion techniques (sitting drop or hanging drop) [66] offer the possibility to manipulate conditions within a protein-containing solution remotely, in this case by diffusion through air. 20

Vapor diffusion

Supersaturation

Initial solution

Final solution

Fig. 4.3 Crystallization by vapor diffusion. In this crystallization method, unsaturated precipitantcontaining protein solutions (indicated on the phase diagram by an open circle) are suspended over a reservoir. Through-vapor equilibration of the droplet and reservoir causes the protein solution to reach a supersaturation level where nucleation and initial growth occur (solid circle). Changes in soluble protein concentration in the droplet are likely to decrease supersaturation over the time course of the experiment (left-most solid circle). Source: [47].

Unsaturation

[Macromolecule]

In the hanging drop set up a microdroplet of mother liquor is suspended from the underside of a microscope cover slip, which is then placed over a small reservoir containing the relatively concentrated precipitating solution (figure 4.3). Coverslips must be thoroughly and carefully coated with nonwetting silicone to ensure proper drop formation and prevent spreading. Using this method, nucleation usually occurs when the protein and precipitant concentrations increase through dehydration-driven reduction of solution volume caused by equilibration of water vapor from the protein containing droplet to a reservoir solution. The difference in precipitant concentration between the drop and the reservoir solution is the driving force which causes water to evaporate from the drop until the concentration of the precipitant in the drop equals that of the reservoir solution. A major advantage of the vapor diffusion technique is the possibility of affecting the equilibration rate and thus approaching supersaturation more slowly, by varying the distance between the reservoir and the crystallization drop. The equilibration process can also be slowed down by inserting an oil barrier over the reservoir [67], [68]. Further advantage is the ability to alter the composition and/or the concentration of the components in the crystallizing solution during the experiment without having to touch the drop. This can be achieved by either concentration or dilution of the reservoir. However, the method has some disadvantages too: once nucleation conditions are achieved, the solution remains highly supersaturated, so that both nucleation and rapid crystal growth can occur simultaneously. Moreover, during the experiment the conditions are changing continuously, thus leaving the investigator as only option to guess at which conditions crystallization exactly occurs. Vapor diffusion and batch are the most widely used techniques mainly due to the relative ease in which they can be set up in comparison with the other methods and automation has been developed for both vapor diffusion and batch. Analytical studies on the process of crystal growth are far more reliable and reproducible in a batch system than in diffusion experiments. This is, because all the crystallization methods, except for batch, involve a change in conditions throughout the crystallization process. In batch the volume and composition of a sample are known and remain constant.

21

Seeding
Given the fact that ideal conditions for nucleation and growth differ (i.e. higher supersaturation for nucleation than for growth), a logical crystallization strategy involves the separate optimization of these processes. This can be accomplished by seeding, a technique where crystals are transferred from nucleation conditions to those that will support only growth. There are two basic seeding methods, both depicted in figure 4.4. In macroscopic seeding [69], one crystal is transferred from the mother liquor where nucleation and initial growth occur to a less supersaturated solution for continued growth. Prior to transfer, the crystal is usually placed in an unsaturated solution to etch its surface. This partial dissolution procedure removes misoriented macromolecules or other matter whose inclusion may have poisoned the crystal surface. Microseeding methods [70] involve transferring a number of very small nuclei to the growth medium. Seed solutions can be prepared by crushing crystals. Major disadvantages of the seeding method are that separate vessels with different solutions are needed, one has to handle very small crystals and, of course, the protein must have been crystallized before.

Macroseeding
Supersaturation

Nucleation and growth solution

Etching solution

Final growth solution

Microseeding

Unsaturation

[Macromolecule]
Nucleation and growth solution Seed solution Final growth solution

Fig. 4.4 Crystallization by seeding. In macroseeding experiments, a single crystal is transferred to an etching solution, then to a solution optimal for growth. Microseeding differs in that a solution containing many small seed crystals, occasionally obtained by crushing a larger crystal, is transferred to a crystal growth solution. The solid circle on the phase diagram indicates that the nucleation and growth solution are supersaturated and within the labile region. The final growth solution (open circle) is sufficiently supersaturated to support only crystal growth and not nucleation. The unsaturated solution used for etching the crystal surface is indicated by the gray circle. Source: [47].

Fortunately, several crystallization methods have been developed where the nucleation and growth conditions can be manipulated separately while keeping the solution in a single vessel. These methods rely on the ability to either transiently or locally achieve nucleation conditions in a solution otherwise sufficiently supersaturated only for growth.

22

Free interface diffusion

Supersaturation

Initial condition

Final condition

Unsaturation

[Macromolecule]
Fig. 4.5 Crystallization methods that separate nucleation and growth conditions within a single vessel. When protein and precipitant solutions first make contact in free interface diffusion experiments, the local supersaturation is high at the intersolution interface (solid circle). The bulk of the protein solution is unsaturated (open circle). As the protein and precipitant solutions mix, the bulk solution remains sufficiently supersaturated to support crystal growth and prevent crystal dissolution (gray circle). Source: [47].

Free interface diffusion Figure 4.5 shows us that in free interface diffusion [71] nuclei form at the interface between concentrated protein and precipitant solutions when the solutions initially come into contact and diffusive mixing occurs so that regions of the protein solution in the neighborhood of the interface become supersaturated from precipitant penetration. As diffusion goes on, smaller nuclei dissolve while larger ones continue to grow. Specific requirements are that the protein is differentially soluble in the two solvents and that one solution can be layered atop of the other. A problem which may occur, unless the experiment is performed under microgravity conditions, is that the protein crystals may sink into the region with very high precipitant concentration. This may lead to growth that is too fast to obtain good quality crystals. A variant of the previous method is the liquid bridge technique: a microdrop of mother liquor and a drop of the precipitating solution are placed in close proximity on a glass slide. A fine needle is used to draw a thin liquid bridge connecting the two drops so that free diffusion can occur between them. The slide and droplets are then sealed from the air to prevent evaporation. By direct liquid diffusion across the bridge and into the mother liquor, the precipitant induces crystallization. Dialysis Just as with batch methods, the protein concentration remains constant during crystallization by dialysis. However, in this method the solution composition is altered by diffusion of low molecular weight components through a semipermeable membrane (figure 4.6, page 24). In this way a protein may be brought slowly towards its precipitation point by dialysis against a solution of concentrated salt or organic solvent. But also dialysis against distilled water is a very productive method. It relies on the limited solubility of many proteins at very low ionic strength, because proteins need to be surrounded by a cloud of positively and negatively charged ions to be soluble. However, dialysis against distilled water is useful only when it does not induce aggregation or denaturation of the protein. Both bulk (large sample volumes) and microdialysis (small sample volumes, in short glass capillaries or so called dialysis buttons) are possible.

23

Dialysis has several advantages, including the ability to change the protein solution composition accurately any number of times and the fact that, as the differential between concentration inside and outside the membrane decreases, the rate of equilibration decreases. Moreover, the protein concentration remains constant, so that only one parameter varies at a time. This enhances a better insight in the roles of different parameters.

Dialysis

Supersaturation

Initial conditions

Final conditions

Fig. 4.6 Crystallization by dialysis. In this method, the protein concentration remains constant because molecules are confined within a fixed volume. Protein solution conditions are changed by dialysis through a semipermeable membrane. As shown on the left, initially the protein solution is unsaturated (open circle on the phase diagram on the right). Changes in the bulk solution alter the protein solubility to achieve supersaturation levels required for nucleation and growth (solid circle). Source: [47].

Unsaturation

[Macromolecule]

However, sometimes a change in protein concentration is the objective and in that case concentration dialysis is an option. Concentration dialysis takes advantage of the fact that many macromolecules will spontaneously crystallize if sufficiently concentrated at low ionic strength. In this method dialysis is performed against a solution of a strongly water binding, long-chain polymer, often poly(ethylene)glycol 20,000 (PEG 20,000). Special method: containerless crystallization Generally, the area of contact between the crystallizing solution and any solid support (like vessel walls and other parts of the experimental set up) is minimized to reduce surface nucleation and to prevent the formation of showers of microcrystals instead of a few nice, large crystals. The application of oil can determine the contact area between the solution droplet and its supporting vessel to a great extent [72]. It is even possible to eliminate all contact by suspending a crystallization drop between two oils of different densities: one of higher and the other of lower density than that of water and the common crystallizing agents (figure 4.7). The drop floats at the interface, thereby not touching the container walls [73], [74]. Sometimes this is called containerless crystallization.

Low density oil

High density oil

Figure 4.7 Containerless crystallization. A crystallization drop is suspended between two oils: the bottom layer contains high-density oil, the top layer contains low density oil. The drop, which has a density between that of the two oils situates itself at the interface between the two oils thereby not touching the container walls. Source: [73].

24

Special method: heterogeneous and epitaxial nucleation In sharp contrast to the objective of reducing all contact with solid surfaces as much as possible, certain inorganic crystals can be purposefully introduced as heterogeneous nuclei or epitaxial substrates in order to facilitate reliable and controlled nucleation in protein crystal growth experiments [75], [76], [77], [78]. Protein crystals can be induced by heterogeneous nucleation and in some cases by epitaxy (for instance, lysozyme on apophyllite (110)) to nucleate and grow at substantially lower supersaturations than needed for spontaneous nucleation. This may improve crystal quality (due to slower growth) and it might even be hoped that it would eventually permit the growth of crystals of hitherto uncrystallizable proteins. Heterogeneous nucleants may induce nucleation through various mechanisms, which conceivably include modification of the mother liquor through adsorption of specific species, introduction of surface chemistry characteristics producing specific interactions with the solute, presentation of a surface microstructure (e.g. roughness) conducive to nucleation, or through characteristics related to the crystalline lattice. Results indicate that mineral substrates with a close lattice match to the protein have a greater influence on the induction time for nucleation and on the number and properties of the protein crystals formed. Special method: crystallization under microgravity conditions The outcome of some of the aforementioned experimental methods can be enhanced by conducting them in a microgravity environment, as can be found aboard a space shuttle or can be established on earth using very high magnetic fields [79], [80]. Protein crystals are more dense than the bulk crystallizing solution, so that an immediate advantage to crystallization in microgravity is the elimination of crystal sedimentation [81]. Crystals grow suspended in solution at the site of nucleation. Similarly, in microgravity, solutions with different densities (which, for instance, form as a result of solute incorporation at the crystal surface) are not subject to convection, equilibration between solutions is much slower and potentially harmful buoyant convective flows are minimized [82], [83], thus allowing crystals to grow via diffusive transport. It is likely that the stabilization of depletion zones and the creation of local regions of reduced supersaturation around growing protein crystals, both due to diffusion limited transport in the absence of any convection, may provide the primary mechanism for an improvement of protein crystal quality. Normally, convection maintains at the growing crystal interface excessive and unfavorable supersaturation as growth proceeds. Remarkably, some of the advantages of crystallization in microgravity, such as elimination of sedimentation and reduced convective flow, can be achieved under normal gravity conditions too, by crystallization in gels. This technique is described in reference [84].

4.2

Approaches to find crystallization conditions

Until now, everything sounds quite simple. Just prepare a supersaturated protein solution by one of the methods described in the previous section and wait for the crystals to appear. However, the basic problem is finding solution conditions that promote protein-protein interactions that are favorable for crystallization. And this, apparently, is not so straightforward. Often, in the study of a particular protein, it is observed that countless combinations of conditions give rise to no precipitate at all or only amorphous precipitate, while the range of conditions suitable for the growth of nice crystals seems to be very limited. So, the main question is: what are the available approaches to find this restricted range of conditions for each protein? At present, two general approaches can be distinguished [73]: one approach concentrates on studies concerning the fundamental understanding of crystal growth on a molecular level. These studies use a limited set of model proteins and investigate many different aspects of crystal growth, such as the kinetics of the growth process, the properties of proteins that seem to be important for crystallization success, characteristics of the crystals formed, ambient factors affecting the outcome, etc. Such studies are of fundamental importance but since crystal growth is 25

a multiparametric process, it is difficult to draw conclusions and many experiments have to be performed. This does not satisfy the investigators who require more immediate results with proteins other than the standard models. Hence, the second approach focuses on empirical methods for finding the right conditions to produce nice crystals. The empirical approach (also known as trial-and-error approach) amounts to searching, as systematically as possible, for the best set of parameters for the crystallization of a particular protein. Various strategies have been set forth for this initial screening of conditions. These vary from highly regimented approaches (like successive grid screening) to analytical approaches (like incomplete factorials, solubility assays, perturbation) to randomized approaches (like sparse matrices). In fact one has the choice between systematically defining and sampling the most important variables that determine whether crystals nucleate ([1], [61], [85], [86], [87]) or applying in a more arbitrary way various screening procedures and sets of crystallization conditions that have proven successful in the past ([88], [89], [90], [91]). Unfortunately, most of these screens, some of them described in more detail below, have as most serious shortcoming that they offer rather little opportunity to integrate the investigators knowledge of the macromolecules physical and chemical behavior into the search for crystals. The incomplete factorial approach [88] is basically a method that, given a matrix of compositional components and their concentrations, defines how to sample the variables with a minimum number of experiments. Using statistical methods to analyze the results, it is possible to identify variables that are correlated, and in later stages to concentrate on their variation to optimize crystallization conditions. An alternative approach is the successive automated grid search method [85]. This method involves the systematic variation of two major crystallization parameters, mostly pH and precipitant concentration. The variation of solution pH and precipitant concentration effectively varies molecular charge as a function of protein supersaturation in searching for suitable crystallization conditions. One can also use sparse matrices [89]. This approach is based on a sparse set of conditions for initial crystallization trials based on conditions which were known to have crystallized proteins. The idea is to provide a broad enough sampling of parameter space by random combination of conditions to yield initial crystals which then may be improved upon. Recently, another, new guide in the search for suitable crystallization conditions was developed, the second osmotic virial coefficient, hereafter referred to as B22 and defined as [92], [93]:

This coefficient is a measure of intermolecular attraction (interaction energy U<0) or repulsion (U>0) averaged over various spatial (and temporal) mutual positions of molecules, their centers being separated by the distance r. NA is the Avogadro number, M is molecular weight. The virial coefficient can be experimentally determined by static light scattering [94]. For the specific case of protein solutions, B22 is a measure of two-body (protein-protein) interactions in a dilute solution condition. It includes contributions from the excluded volume term, electrostatic factors and hydrophobic interactions. A solution condition that corresponds to a positive value for B22 generally indicates strong repulsive forces between the protein molecules. In that case, the protein-solvent interactions are likely to be favored over protein-protein interactions and thus no crystal formation occurs. Strong attractive forces between protein molecules give rise to large negative values for B22. In general, these solution conditions produce nonspecific aggregates and eventually an amorphous solid phase. The event of crystallization seems to take place only in solvents which provide a moderately negative virial coefficient, situated in the so called crystallization slot, containing B22 values in the range of (2-8) x 10-4 mol*ml/g2. This range of B values promotes the formation of microcrystal22

line aggregates at high enough protein concentrations while limiting the probability of nonspecific 26

aggregate formation. The existence of such a limited B22 range favoring crystallization is supported by the data in figure 4.8. These findings may be understood as a possibility for the system, on the one hand, to have sufficiently strong specific chemical intermolecular attractions to carry on crystallization and, on the other hand, to have this attraction weak enough to allow selection of species in proper orientation. A very important point regarding the crystallization slot is that it is achieved for a broad range of different proteins with many different crystallizing solvent conditions. A practical example of the use of the second osmotic virial coefficient for the prediction of conditions favorable for the crystallization of chymotrypsinogen can be found in [17].

Fig. 4.8 Crystallization slot. The crystallization slot represents a range of osmotic second virial coefficient, B22, values that were measured for about 20 different crystallization conditions for a variety of proteins and crystallizing agents. Source: [1].

27

Chapter 5 Nucleation and growth processes of protein crystals

5.1 Methods of investigation

A large body of methods exists to investigate prenucleation, nucleation and crystal growth processes of proteins. These are listed in the following table.
Table 5.1 Physical approaches for studying macromolecular crystallization. Source: [1].

Method
Static/quasi-elastic light scattering Michelson interferometry Mach-Zehnder interferometry Atomic force microscopy Fluorescence polarization Low angle neutron scattering Osmometry Light microscopy Time lapse video microscopy X-ray diffraction Numerical simulation/modeling

Use
Nucleation, phase transitions Growth kinetics Concentration gradients in solutions Growth mechanisms, kinetics, defect structure, defect density Nucleation Nucleation Nucleation Characterization of crystals Growth kinetics Characterization of crystals Nucleation, growth kinetics, phase transitions

Investigations based on light scattering have been most informative in delineating prenucleation processes and structures [4], [95], [96]. The idea was to understand the nature of prenuclear aggregates and clusters that form in solution and how they transform into crystal nuclei. Interferometry and atomic force microscopy (AFM) are powerful techniques to qualitatively study surface morphology and crystal growth processes in situ. Furthermore, with these methods it is possible to quantize some of the parameters that govern the crystal growth processes, such as the step free energy and the kinetic coefficient for step motion [1]. The foundation of this chapter is formed by papers about investigations using the abovementioned techniques. With AFM the growth mechanisms of canavalin [97], [98], [99], [100], thaumatin [99], [100], [101], [102], apoferritin [99], catalase [99], [100], [101], [103], lysozyme [99], [101], [104], xylanase [100], [101] and lipase [100] were studied. Of canavalin [105], thaumatin [105] and lysozyme [106], [107] also useful studies with in situ laser Michelson interferometry were published. Further information was extracted from a couple of reviews [1], [4], [6], [12], [15], [18], [54], [107], [108], [109] and an article about theoretical models for protein nucleation and crystal growth [5]. In the following text, I will further provide you with a couple of other references to somewhat more specific aspects of protein crystal growth and sometimes I will point you to one of the references mentioned here.

5.2

Formation of critical nuclei

A good overview of results on nucleation and of current nucleation theories can be found in [1]. The nucleation of globular proteins is governed by the same principles as that of conventional , small molecules. In the nonequilibrium state of supersaturation, molecules continually associate to form clusters and roughly spherical aggregates of varying order. If, at some point, the dimensions of the ordered aggregate reach a size such that the radius exceeds a certain critical value, then it will accumulate new molecules more rapidly than it will lose old members and one can speak of a real crystal nucleus. Estimates of the critical nucleus as a function of supersaturation have been obtained by quasi-elastic light scattering. For proteins the critical nucleus sizes are similar to the ones of conventional molecules, but one to two orders of magnitude less in terms of the numbers of particles incorporated. 28

Fig. 5.1 Various pathways for the spontaneous formation of a critical nucleus in solution. (A) Linear aggregates of molecules become branched by adhesion to one another and by the formation of forks followed by the addition of monomers. The fractile-like structures then fold and collapse upon themselves to from threedimensional aggregates of critical size. (B) Threedimensional ordered aggregates form by the sequential ordered addition of monomers, while in C, the same process occurs by the sequential ordered addition of some specific oligomer, in this case dimers. (D) Ordered aggregates are formed directly, with no restructuring, by the mixed, ordered addition of monomers and various oligomers. Pathway E, is the fluid aggregate model, which depends on the formation of an ordered region within a large random aggregate that subsequently propagates to become critical size. Source: [1].

At present, the nature of the nuclei and the processes by which they form are largely unknown, despite extensive investigations with the techniques mentioned above. Whether the aggregate that forms the critical nucleus is ordered from the beginning or assumes order through a restructuring is not certain, nor is it certain whether it forms by the coalescence of arbitrary subnuclear clusters or by strict monomer or multimer addition, or by all of these in parallel. Some models of plausible nucleation pathways are illustrated in figure 5.1, but experimental observations are still insufficient to decide which of these models correlates best with reality. Moreover, different proteins may follow different nucleation pathways. However, one of the nucleation models, the so called fluid aggregate model (figure 5.2), is especially appealing, because it incorporates cooperative processes of the kind that characterize many biochemical and biophysical phenomena. The model takes advantage of the inherent fluidity of collective weak interactions, the dynamic structure of their arrangements and their ability to transition en mass from disordered to ordered states. Proteins have the capability, and indeed are known, to form more or less nonspecific aggregates as a consequence of hydrophobic interactions and random hydrogen-bonding networks. Such aggregates almost certainly contain a large number of mobile water molecules as well. Rearrangement of the solvent could facilitate an ordering process in a fluid aggregate that ultimately leads to a more periodic and stable bonding network. In later stages this might promote the formation of more geometrically demanding salt bridges and specific hydrogen-bonding networks leading to a true crystal nucleus.
Fig. 5.2 Fluid aggregate model. As the supersaturation is increased and association of molecules is promoted, molecules begin organizing into large disordered aggregates (I). The interactions are principally nonspecific, transitory, hydrophobic interactions that allow the molecules motion and flexibility. With time, molecules within the cores of such aggregates, more or less protected from solvent, reorient, redistribute and give way to more geometrically rigorous hydrogen bonds and electrostatic interactions (II). The formation of these latter interactions tend to order and stabilize the aggregate core which subsequently increases in size to produce a critical nucleus (III). This ultimately develops into a true crystal (IV). Free molecules are then adsorbed to the crystal surface and increase its size by their incorporation into its lattice (V). Source: [1].

29

Whether protein crystal nucleation can proceed by this mechanism remains to be seen. Certainly, however, many investigations support the idea that the formation of prenucleation aggregates might play a considerable role in crystal formation. For instance, besides the experimental lightscattering evidence of the existence of many aggregates in protein solutions, Salemme et al. observed that several different crystal forms of the same protein can be viewed as the result of alternative ways of assembling the same primitive linear chains of protein molecules [20]. Therefore, aggregated chains might form as precursors to these crystals.

5.3

Growth mechanisms, the growth unit, transport processes and kinetics

Overviews of protein crystal growth mechanisms and kinetics can especially be found in [1], [4], [6], [12], [18], [54], [108] and [109]. Results on several proteins in particular can be found in the references enumerated at the beginning of the chapter. It appears that, in spite of the structural complexity of protein molecules as well as the compositional complexity of protein solutions (that is the presence of precipitant and pH-buffer components), there are pronounced similarities between the mechanisms and kinetics underlying the crystallization of proteins and inorganic materials. Growth mechanisms On a macroscopic scale, most protein crystals have well defined, flat facets. This unambiguously implies that the crystals grow layer by layer, with each layer spreading laterally more rapidly than a new one forms on top of it. AFM observations on a large number of proteins have reproduced the whole body of growth mechanisms known for conventional inorganic solution growth. These include layer spreading from screw dislocations and two-dimensional nuclei and also the mechanism of normal or roughened growth, characterized by intensive random nucleation. However, in protein crystal growth there appears to be an additional mechanism that involves the ordered addition of large three-dimensional aggregates. It has been shown that individual macromolecular crystals may simultaneously employ different mechanisms on the same face [10] or transform from one mechanism to another as a function of supersaturation [110]. In the case of spiral dislocation growth on protein crystals, usually multiple dislocation sources generate steps without any one dislocation becoming dominant. Both single and double, left- and right-handed and multiple interacting spirals have been observed (figure 5.3) [111], [112].

Fig. 5.3 Spiral dislocation growth. In situ AFM images show single and double dislocations on the surfaces of canavalin and lysozyme crystals. (A) a single spiral dislocation on the (100) face; (B) a pair of interacting, double, right-handed spiral dislocations; (C) a complex dislocation source containing three interacting single dislocations; (D) A double growth spiral on the (110) face of a tetragonal lysozyme crystal. The scan areas for the AFM images are 30x30 (A), 22x22 (B),12x12 (C) and 6x6 (D) m. Source: [1].

30

In spite of the fact that spiral dislocations are occasionally observed at low supersaturations, twodimensional nuclei are clearly the dominant sources for growth steps over the entire supersaturation range, even near equilibrium, for most proteins [111]. On small crystals, two-dimensional nucleation sites are randomly distributed over the faces. With increasing supersaturation/growth rate and facet size, nucleation preferentially occurs along the crystal edges and, eventually, facet corners. This is caused by an increase in nonuniformity of solute supply from the bulk, as the crystal size and growth rate increase, leading to a higher interfacial supersaturation closer to the crystals edges. Yet, despite this supply nonuniformity, macroscopic flatness of the facets is retained in many cases. This macroscopic morphological stability arises from nonuniform changes in the local kinetics parameters (step densities) associated with the changes in the microscopic morphology that forms in response to the solute nonuniformity.
Fig. 5.4 Normal growth. In situ AFM images of apoferritin crystals in A and B. Surfaces of apoferritin crystals are atomically rough and normal growth was observed. The surfaces are consistently extremely irregular and rough, as is characteristic of crystals that arise by a normal growth mechanism. Source: [1].

Normal growth is not often observed, one of the few cases is in the crystallization of apoferritin (figure 5.4) [99], [111]. The observed roughness of the surfaces of apoferritin crystals is caused by intensive random nucleation at high supersaturation conditions, a phenomenon known as kinetic roughness. Macromolecular crystal growth proceeds by at least one apparently unique mechanism: growth by direct addition and subsequent development of intact, three-dimensional nuclei, in many cases microcrystals [100]. Upon adsorption, the three-dimensional nuclei proceed to grow in both the normal direction, by two-dimensional nucleation on their own faces, and by step flow over the surface of the larger, growing crystal. The most remarkable feature of this phenomenon is that the lattices of the three-dimensional nuclei merge and knit with the lattice of the larger crystal to which they adsorb, without creating defects or dislocations. It suggests that the underlying lattice either guides crystalline nuclei into preferred orientations as they adsorb, or that some restructuring of otherwise disordered aggregates, guided by the underlying lattice results in alignment. The aggregates are inferred to originate from liquid-protein droplets based on the phase diagram of a protein-water system, which shows a metastable liquid-liquid immiscibility region [113], [114]. Below a certain critical temperature there are two coexisting liquid phases and small liquid droplets with a high protein concentration are formed. Either small crystalline nuclei or aggregates may form inside these droplets. In figure 5.5 (on the next page) the above described and an alternative mechanism for the formation of three- and two-dimensional nuclei on a crystal surface are schematically depicted. The growth unit Protein crystal growth involves the incorporation of a complex unit into an existing lattice. The growth unit usually includes the covalent polypeptide chain, water molecules that are integral components of the folded protein structure and additional water molecules and solvent ions that may become immobilized at crystal lattice contacts. As stated before, from light scattering experiments it is known that proteins can form (transient) aggregates in solution. This gives rise to the question whether the growth units are monomeric protein molecules or aggregate forms, consisting of several molecules. 31

There appear to be a few specific proteins with aggregate growth units, e.g. insulin [54], [92]. In these proteins the charged groups on the surfaces occupy positions that leave a large number of hydrophobic side chains open to contact with water. On aggregate formation, the hydrophobic patches on the monomers are covered (by other protein molecules) and therefore, these proteins show a strong tendency to aggregation prior to incorporation. There has also been speculation that the growth of tetragonal lysozyme crystals may occur through the incorporation of tetramers or octamers that form in the solution prior to attachment to the crystal [104], [116]. However, compared to, par example, insulin, the hydrophobic surface area of the lysozyme monomer is much smaller and thus the tendency to form aggregates is strongly reduced. Indeed, careful static and dynamic light scattering investigations of the lysozyme solutions from which the crystals grow have not revealed the presence of any species other than the lysozyme monomer [54], [117], [118]. Thus, the addition of ordered clusters of fixed size, random size or disordered aggregates may occur for a couple of proteins with specific surface properties, but for most proteins it is more probable that growth step advance occurs by incorporation of single protein molecules.

Fig. 5.5 Formation of two- and three-dimensional nuclei. A schematic diagram illustrating how two- and three-dimensional nuclei, giving rise ultimately to two-dimensional islands and multilayered stacks of growth layers, may appear on the surfaces of growing macromolecular crystals. In A, monomers of the protein associate in a sequential manner on the crystal surface to form a two-dimensional island. Liquid protein droplets, having short-range internal order, sediment on the surfaces and are instructed by the underlying lattice (an epitaxial mechanism), how to arrange themselves in a manner that is consistent with the basal lattice in B and C. Because the clusters or fluid aggregates are locally hypersaturated, the crystallization, which is promoted initially at the interface with the substrate crystal, propagates upwards layer by layer. Small aggregates give rise to two-dimensional islands (B), while larger aggregates develop into large multilayered stacks (C). Source: [1].

32

Transport processes and kinetics In order to be incorporated, a growth unit has to be transported to the growing crystal surface. Transport includes pure diffusion of the molecules, convective transport through solutal flow and surface diffusion after adsorption to a face. Unless conducted between very closely spaced solid boundaries (such as microscope slides a few tenths of a millimeter apart) on earth, protein crystallization will always be associated with convective transport that typically dominates diffusive fluxes. This is because gravity can act on the density difference between the solute-depleted solution layer near the growing crystal surface and the bulk solvent to produce convection currents. Based on experimental observations it may well be assumed that solute transport to steps is a multistage process involving bulk transport (diffusion/convection), adsorption on the interface and surface diffusion toward the steps (see also the model presented at the closing of this section). It appears to be that surface diffusion rather than bulk transport is the controlling mechanism of solute transport to the steps [119]. However, crystal growth rates do not only depend on the transport rate of solute molecules to the steps, but also on their rate of incorporation after they have arrived. Most studies suggest that such surface effects are rate limiting in protein crystal growth, that is to say that the growth of proteins is limited by kinetics rather than transport. Important effects occurring at the lattice surface include the formation of favorable growth sites (usually kinks) and molecular attachment to these sites. Crystallization of biomolecular and inorganic crystals from solutions obey the same kinetic laws, so in the study of protein crystal growth kinetics we can use the same fundamental parameters: the free energy of molecular addition at the step edge, , and the kinetic coefficient, . The free energy of molecular addition provides a measure of the work required to increase the surface of a crystal by one unit. The kinetic coefficient is a measure of the kinetics of adsorption, diffusion and incorporation, with the rate limiting step dominating its value. From the dimensions of nuclei greater than critical size and also from rates of step advancement, it has been possible to calculate the free energy of the step edge and the kinetic coefficient for the growth of several protein crystals [97], [99], [101], [102], [105], [107], [111], [120], [121], [122], [123], [124]. Typical values are (0.4 - 2.4) x 10-8 J/cm2 for and (0.3 12) x 10-4 cm/sec for . These values are two to three orders of magnitude less than for conventional crystals, however, in the case of , if one considers the surface area of a protein molecule then the total energy required to incorporate it into a crystal is about the same as for a small molecule. The potential source for the low value of the kinetic coefficient , indicating slow step advancement and thus low growth rates, include a large barrier to adsorption (shedding of the hydration sphere), low surface diffusivity and a low probability that an incoming molecule has the proper orientation for incorporation into the crystal. The latter, which is the major qualitative difference from simple inorganic species, is often indicated as the steric probability factor. This factor gives the probability that a molecule arrives at the kink position possessing the correct spatial orientation to allow incorporation into the lattice, relative to all other orientations. The steric probability factor for macromolecules is on the order of 10-2 10-3 [15] compared with values close to unity for small, inorganic molecules. This is understandable, because for small inorganic species, one may expect that the electrostatic field of the lattice and the lattice electron clouds penetrating into the solution in the kink vicinity may rotate the approaching species to the proper orientation. With proteins the field also extends over distances comparable to the atomic dimensions. However, now, this is much less than the molecular size. The charges on the molecular surface are screened, though not completely, by precipitant salt ions down to atomic scale. Therefore, the rotation moment should be small and a molecule may be properly incorporated only if it arrives at the kink having the orientation within the range of spatial angles where atomic forces are still able to rotate the molecule into a further more precise orientation. 33

Usually, protein crystals display a strong kinetic anisotropy in step advancement [40], [101]. This means that for steps moving in different crystallographic directions on the same crystal surface the advancement rates, and thereby the kinetic coefficients , can differ to a great extent. This leads to asymmetric shapes of expanding growth spirals and two-dimensional islands (figure 5.6) Step anisotropy arises because of differences in molecular structure of growth steps advancing in different crystallographic directions. Molecules forming step edges moving in different crystallographic directions expose different parts of their surfaces. This results in different distributions of charges, hydrogen bond donors and acceptors and hydrophobic clusters on the step edges, as well as differential screening by ions. This explains the different rates of molecule incorporations into step edges advancing in different directions.

Fig. 5.6 Growth anisotropy. Asymmetric spiral dislocations are seen here on the developing surfaces of crystals of thaumatin (A), canavalin (B) and xylanase (C). (D) Strikingly asymmetric, twodimensional islands appear on the surface of a growing catalase crystal. Source: [1].

Growth studies indicate that growth variables can fluctuate by as much as ~ 80% of their average values [125]. Many fluctuations are due to the passage of step bunches, i.e., the steps on the interface are not equidistant but rather grouped into a pattern of lower and higher step density. Such variations may arise from the intrinsically irregular nature of step generation by either two-dimensional nucleation or complex dislocation sources. Further irregularities in the step motion, and hence step density, may be introduced by various obstacles that impede the steps progress: adsorbed impurity molecules or larger particles, point defects, dislocation outcrops and other surface imperfections. In reference [101] records of experimental observations of step bunching can be found. Although the knowledge of protein crystal growth still is far from complete, as a conclusion of this section a tentative model for the growth of protein crystals is presented. From a very simple point of view crystal growth essentially consists of merely two processes: transport and incorporation of growth units. Now that we have identified the important stages of crystal growth, we can refine it further leading to the model described below and illustrated in figure 5.7 (page 35). However, it may, as presented, lack events that are important to the process, and on the other hand, it may contain some that are irrelevant. In crystal growth a molecule goes through the following steps: (1) diffusion from bulk solvent to surface layer or interface, (2) diffusion through this surface layer and (3) collision with and perhaps adsorption to the crystal surface. Adsorbed to the crystal a molecule must (4) move by twodimensional diffusion over the surface until it reaches a potential incorporation site. Once a molecule has reached the incorporation site, it must (5) undergo rotational diffusion until it achieves an orientation that allows it to form stable, but still fluid bonds with molecules that are already present in the lattice. During or following these translational and rotational processes, a molecule may (6) be obligated to undergo partial dehydration or rearrangement of its hydrating water molecules and possibly (7) experience conformational changes to conform to the lattice building units. The molecule (8) may form quasi-stable but still fluid bonds with molecules in the lattice, perhaps involving van der Waals or transient hydrogen bonds. Bonds then may undergo rearrangement (9) to form the final crystalline bonding arrangement, which may be accompanied, in addition, by (10) liberation of additional water molecules. Finally, the stable incorporation of a molecule into a site may require (11) the establishment of a water network between the new recruit and the previous members of the ensemble, as well as with the local water environment. 34

Fig. 5.7 Protein crystal growth model. A model for the growth of macromolecular crystals from monomers (I and II) or ordered aggregates, here trimers, from solution (III), where the crystallizing species either diffuses across the surface of the growing crystal (I) or incorporates directly into a lattice site (II and III). In (I), the molecule must be transported to the growing crystal surface, (A) adsorb, (B) diffuse across the surface, (C) undergo conformational changes, (D) undergo rearrangements of water molecules, and (E) finally be incorporated at a lattice point. Even with direct incorporation, as in (II) and (III), a similar series of events may transpire. We cannot be certain of all of the steps involved or their significance to the mechanism and kinetics of the process. Source: [1].

35

Chapter 6 General discussion and conclusion

Clearly, the crystallization of proteins is a highly interesting field of research, because of its outstanding performance as a model system for crystallization from solution in general and because of the amazing amount of useful information that can be obtained about the structure of a protein molecule by X-ray diffraction analyses of its crystals. Unfortunately, however, in most cases the crystallization of a protein is not an easy job to do. Partly, this is due to the complexity of protein molecules and their properties, their sharp demands on their environment and the extensive interactions between environmental and protein properties. But it is also due to a lack of fundamental knowledge about the processes at work. During the course of protein crystal growth history, a whole body of crystallization methods has been developed. However, theoretical insight in the underlying principles is still lagging sharply behind. This may be caused by the aforementioned complexity of the process, but my opinion is, that it is also to a large extent due to the point of view of most investigators at work in this field. Most researchers want crystals of proteins, to get to know more about their structure, in a time span as short as possible. Consequently, they use an empirical trial-and-error approach that is purely directed to rapidly finding suitable crystallization conditions for a particular protein, without bothering too much, if bothering at all, about the underlying principles. These approaches surely are not directed to obtaining more insight in the general crystallization processes at work and also do not stimulate the initiation of any (more time consuming) fundamental research on protein crystallization in general. Maybe, if no crystallization screens had been developed, there would have been much more interest in this type of research, because in the long run it will undoubtedly provide opportunities to use a much more directed approach. In such an approach tailor made crystallization methods and conditions could be used for each particular protein, based on its specific properties, leading to a much higher rate of success. At present, only tentative ideas exist about interactions between proteins, the relation between the properties of a protein molecule and its behavior in solution or during crystallization, the parameters that play an important role in crystallization and the nature of nucleation processes, crystal growth mechanisms and kinetics. If we ever want to be really successful in protein crystallization we have to gain much more insight in each of these, amongst many other, aspects. In order to achieve this insight we will have to concentrate on studies concerning the fundamental understanding of crystal growth on a molecular level. For instance, this means that we will have to perform elaborate studies on the influence of separate parameters. In this process of investigation, I want to propagate a high degree of open-mindedness, as the majority of research has too long been based on the known-to-be-good principle. Par example, take a look at nonconventional parameters nobody has thought of until now and do not exclude possibilities to early. Maybe, the approach suggested here seems to have no sound basis, but then it is good to remember that there appear to be pronounced similarities between the mechanisms and kinetics underlying the crystallization of proteins and conventional inorganic materials, in spite of the much greater structural complexity of protein molecules as well as the compositional complexity of protein solutions. Therefore, contrary to the past, one should make more use of the very thorough theoretical knowledge of conventional crystallization systems. This may lead to many new insights. I want to conclude with a practical advise for anybody who is planning to do fundamental investigations: use an experimental set up offering as much simplicity and controllability as possible. In this context, it might be a good idea to explore the possibilities of temperature induced crystallization. In conventional systems, temperature is known to be a very suitable parameter to control supersaturation. It is relatively easy to control, you do not have to alter the solution composition in order to reduce the proteins solubility and it is not necessary to add large amounts of additives. The latter means that the solution composition will be far less complex and this might make the understanding of the processes at work much less complex too. 36

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