Structural Biochemistry/Proteins/Structures

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Contents
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1 Primary Structure
o 1.1 Forces that stabilize Protein Structure
o

1.2 Factors that cause denaturing

1.3 Determination of Primary Structure: Amino Acid Sequencing

2 Secondary Structure
o

2.1 Alpha Helix

2.1.1 Structure

2.1.2 Supersecondary Structure of Alpha Helix

2.1.2.1 Fibrous Proteins

2.1.3 Alpha Tertiary

2.1.4 SUMMARY

2.2 Beta Pleated Sheet

2.3 Turn and Loop

2.4 Beta Hairpin Turns

2.5 Fibrous proteins

2.6 Secondary Structure Prediction

2.6.1 Torsion Angles

2.6.2 Ramachandran Diagram

3 Tertiary Structure
o

3.1 Structure

3.2 Domains

3.3 Mutations

3.4 Folding

3.5 Determination of Tertiary Structure

4 Quaternary Structure
o

4.1 Naming Quaternary Structures

4.2 Dimers

4.3 Trimer

4.4 Tetramer

4.5 Breaking Apart the Quaternary Structure

4.6 Protein Folding

4.7 Analogy

4.8 Human attempt to manipulate protein assemblies (Quaternary Structures)

Primary Structure
The primary structure of a protein is a linear polymer with a series of amino acids. These amino
acids are connected by C-N bonds, also known as peptide bonds. The formation of peptide bonds
produce water molecules as a by-product when an amino terminal residue (N-terminal) loses an
oxygen from the alpha-carboxyl group while the other amino acid loses two of its hydrogens
from its alpha-amino group. Thus, polypeptide, or polypeptide chain, is a term that describes the
multiple connected peptide bonds between numerous amino acids. Each amino acid in a
polypeptide chain is a unit, commonly known as a residue. These chains have a planar
backbone, as the peptide bonds have double bond characteristics due to the existence of
resonance between the carbonyl carbon and the nitrogen where the peptide bonds form. The
primary structure of each protein has been precisely determined by the specific genes. The C-N
bond in an amino acids chain has the character of a double bond. This bond has a short length
and stable. It cannot be rotated. This double-bond character can be explained structurally, in that
the R groups in amino acid chains avoid steric clash.
Amino acids are linked by peptide bonds to form polypeptide chain; each amino acid unit is
known as a residue; a polypeptide chain constructed by the same unit is known as the main chain
or backbone and a changing R group, side chains.

Forces that stabilize Protein Structure

Protein structures are governed primarily by hydrophobic effects and by interactions

between polar residues and other types of bonds. The hydrophobic effect is the major
determination of original protein structure. The aggregation of nonpolar side chains in
the interior of a protein is favored by the increase in Entropy of the water molecules
that would otherwise form cages around the hydrophobic groups. Hydrophobic side
chains give a good indication as to which portions of a polypeptide chain are inside,
out of contact with the aqueous solvent. Hydrogen bonding is a central feature in
protein structure but only make minor contributions to protein stability. Hydrogen
bonds fine tune the tertiary structure by selecting the unique structure of a protein
from among a relatively small number of hydrophobically stabilized conformations.
Disulfide bonding can form within and between polypeptide chains as proteins fold to
its native conformation. Metal ions may also function to internally cross link proteins.

Factors that cause denaturing

1)Temperature
2) pH
Extreme temperatures will result in the unfolding of a polypeptide chain leading to a
change in structure and often a loss of function. If the protein functioned as an enzyme
denaturing will cause that protein to lose its enzymatic activity. As the temperature of
a solution containing the protein is raised, the extra heat causes twisting and bending
of bonds. As proteins begin to denature the secondary structure of the protein is lost
and adopts a random coil configuration. Covalent interaction between amino acid side
chains such as disulfide bonds are also lost.
At high or low pH levels the protein will denature due to the lose or gain of a proton
and, therefore, will lose their charge or become charged, depending on which way the
pH is changed and by how much. This will eliminate many of the ionic interactions
that were necessary for maintenance of the folded shape of the protein. As a result the
change in structure will cause a change or loss of function.

Determination of Primary Structure: Amino

Acid Sequencing
After the polypeptide has been purified, the composition of the polypeptide should be
established. To determine which amino acid and how much of each is present, the
entire strand is degreaded by amide hydrolysis (6N HCl, 110 0C, 24hr) to produce a
mixture of all free amino acid residues. The mixture is separated and its composition

recorded by amino acid analyzer. The amino acid analyzer establishes the composition
of a polypeptide by giving a chromatogram, which records the peaks of each amino
acid presents in the sequence. However, the amino acid analyzer can only give the
composition of a polypeptide, not the order in which the amino acids are bound to one
another.
To determine the amino acid sequence, it usually starts from the determination of the
amino terminal of the polypeptide. The procedure is known as Edman degradation,
and the reagent employed is phenyl isothiocyanate.

Phenyl isothiocyanate
In Edman degradation, the terminal amino group adds to the isothiocyanate reagent to
produce a thiourea derivative. Treating with mild acid, the tagged amino acid is turned
into a phenylthiohydantoin, and the remainder of polypeptide is unchanged. Since the
phenylthiohydantoins of all amino acid are known, the amino terminal of the original
polypeptide can be identified easily. However, Edman degradation can only be used to
identify the amino end of the polypeptides; therefore, for polypeptides that are made
up by hundreds of amino acids, it is not a practical method in general. In addition,
multiple degradation rounds will build up impurities which will seriously affect the
yield of peptide. High yield means not completely quantitative, and with each step of
degradation, incompletely reacted peptide will mix with the new peptide, resulting in
a intractable mixture.

Secondary Structure
Secondary structures of proteins are typically very regular in their conformation. They
are the spacial arrangements of primary structures. Alpha Helices and Beta Pleated
Sheets are two types of regular structures. An interesting bit of information is that
certain amino acids making up the polypeptide will actually prefer certain folding
structures. The Alpha Helix seems to be the default but due to interactions such as
sterics, certain amino acids will prefer to fold into Beta pleated sheets and so on. For
example, amino acids such as Valine, Isoleucine, and Threonine all have branching at
the beta carbon, this will cause steric clashes in an alpha helix arrangement. Glycine is
the smallest amino acid and can fit into all structures so it does not favor the helix
formation in particular. Therefore, these amino acids are mostly found where their
side chains can fit nicely into the beta configuration.

The structure of polypeptide main chains is mostly of hydrogen-bonding; each residue

has a carbonyl group that is a good hydrogen- bond acceptor; nitrogen- hydrogen
group, a good hydrogen- bond donor.

Alpha Helix
Structure
The general physical properties of an alpha helix are:

Alpha helix project outward in helical array

Ribbon displaying the backbone of the alpha helix

3.6 residues per turn
Translation (rise) of 1.5 A

Rotation of 100 degrees

Pitch (or height) of 5.4A (1.5A*3.6 residues)

Alpha helix with hydrogen bonds

Screw sense = clockwise (usually) because it would be less sterically hindered
Inside the helix consist of the coiled backbone and the side chains project
outward in helical array

Hydrogen bonding between the 1st carbonyl to the hydrogen on the 5th amino

The shorthand drawing of the alpha helix is a ribbon or rod

Ribbon shorthand notation for the alpha helix

Ramachandran diagram
Alpha helix falls within quadrant 1 (left-handed helix) and 3 (right-handed
helix) in the Ramachandran diagram

Supersecondary Structure of Alpha Helix

Fibrous Proteins
I. COILED-COIL (-keratin)
An alpha coiled coil consists of two or more alpha helices intertwined, creating a
stable structure. This structure provides support to tissues and cell, contributing to the
cell cytoskeleton and muscle proteins such as myosin and tropomyosin. Alpha keratin
consists of heptad repeats (imperfect repeats of 7 amino acid sequences). This
facilitates bonding between the two or more helices.
II. COLLAGEN
Collagen is another type of fibrous protein that consists of three helical polypeptide
chains. It is the most abundant protein found in mammals, making up a large
component of skin, bone, tendon, cartilage, and teeth. Wrinkles are also caused by the
degradations of this protein. In the structure of collagen, every third residue in the
polypeptide is glycine because it is the only residue that is small enough to fit in the
interior position of the superhelical cable. Unlike normal alpha helices, each collagen
helix is stabilized by steric repulsion of the pyrrolidine rings of the praline and
hydroxyproline residues. However, the three strands intertwined are stabilized by
hydrogen bonding.

Alpha Tertiary
I. MOTIFS
Motifs are simple combinations of the secondary structure such as the helix-turnhelix, which consist of two helices separated by a turn. The helix-turn-helix motif are
usually found in DNA-binding proteins.
II. DOMAINS (GLOBULAR)
Domains, or compact globulars, consist of multiple motifs.They are polypeptide
chains folded into two or more compact regions connected by turns or loops. Their
structure is spherical, which is beneficial for the protein because it conserves space.
Generally, inside the globular protein consist of hydrophobic amino acids such as
leucine, valine, methionine, and phenylalanine. The outside consists of amino acids
with hydrophilic tendencies such as aspartate, glutamate, lysine, and arginine. An
example of a globular protein is myoglobin, which is the oxygen carrier in muscle. It
is an extremely compact molecule made of only alpha helices (70%) except for loops
and turns (30%).

SUMMARY
The alpha-helix consists of a single polypeptide chain in which the amino group (NH) hydrogen bonds to a carboxyl group (C=O) 4 residues away. The alpha - helix is a
rod-like structure. The tightly coiled backbone of the chain forms the inner part of the
rod and the side chains extend outward in a helical array. This results in a clockwise
coiled structure, which is known as a "right handed" screw sense. This folding pattern,
along with the beta-pleated sheets were actually proposed by Linus Pauling and
Robert Corey half a decade before people could actually see it. Most of the alpha
strands are located in the lower left corner or upper right corner of the Ramachandran
diagram . Essentially, most of the alpha helices are found in the right-hand helices
area. An alpha helix is especially suited for cross-membrane proteins because all of
the amino hydrogen and carbonyl oxygen atoms of the peptide backbone can interact
to form intrachain hydrogen bonds while its aliphatic side chains can stabilize in
hydrophobic environment of cell membrane.
Alanine, leucine and glutamic acid (existed as glutamate as physiological pH) are the
most common residues present in alpha-helices.
The alpha-helix content of protein ranges widely, from none to almost 100%.
In general, the alpha helix is the "normal" shape of a polypeptide chain; however,
features of certain amino acids disrupt alpha helix formation and instead favor beta
strand formation. Amino acids with branching at the beta carbon (i.e. valine,
threonine, and isoleucine) are problematic because they crowd the peptide backbone.
H-bond accepting/donating groups attached to the beta carbon (i.e. serine, asparagine,
and aspartate) can bond with backbone amine and carboxyl groups, again interfering
with alpha helix formation.
While individual amino acids may favor one form or another, predicting the 2
structure of even a short (<7 amino acid) peptide strand is only 60-70% accurate. Such
variability suggests other factors, like tertiary interactions with amino acids further
down the chain, influence the folding into its observed 3 structure.

Beta Pleated Sheet

In contrast to the alpha helical structure, Beta Sheets are multiple strands of
polypeptides connected to each other through hydrogen bonding in a sheet-like array.
Hydrogen bonding occurs between the NH and CO groups between two different
strands and not within one strand, as is the case for an alpha helical structure. Due to
its often rippled or pleated appearance, this secondary structure conformation has been

characterized as the beta pleated sheet. The beta strands can be arranged in a parallel,
anti-parallel, or mixed (parallel and anti-parallel) manner.

Anti-parallel Beta Strand

The anti-parallel configuration is the simplest. The N and C terminals of adjacent
polypeptide strands are opposite to one another, meaning the N terminal of one
peptide chain is aligned with the C terminal of an adjacent chain. In the anti-parallel
configuration, each amino acid is bonded linearly to an amino acid in the adjacent
chain.

Parallel Beta Strand

The parallel arrangement occurs when neighboring polypeptide chains run in the same
direction, meaning the N and C terminals of the peptide chains align. As a result, an
amino acid cannot bond directly to the complementary amino acid in an adjacent
chain as in the anti-parallel configuration. Instead, the amino group from one chain is
bonded to a carbonyl group on the adjacent chain. The carbonyl group from the initial
chain then hydrogen bonds to an amino group two resides ahead on the adjacent
chain. The distortion of the hydrogen bonds in the parallel configuration affects the
strength of the hydrogen bond because hydrogen bonds are strongest when they are
planar. Therefore, due to this distortion of hydrogen bonds, parallel beta sheets are not
as stable as anti-parallel beta sheet (exp: formation of parallel beta sheet with less than
5 residues is very uncommon).
The side chains of beta strands are arranged alternately on opposite sides of the strand.
The distance between amino acids in a beta strand is 3.5A which is longer in
comparison to the 1.5A distance in alpha strands. Because of this, beta sheets are more
flexible than alpha helices and can be flat and somewhat twisted. The average length

of beta sheets in a protein is 6 amino acid residues. The actual length ranges from 2 to
22 residues.

Ramachandran Plot: Beta strands are found in the purple region

Beta sheets are graphically found in the upper left quadrant of a Ramachandran plot.
This corresponds to angles of 0 to 180 and angles of -180 to 0.

The schematic model of beta sheets

Visual representations in 3D models for beta sheets are traditionally denoted by a flat
arrow pointing in the direction of the strand.

Turn and Loop

Polypeptide chains can change direction by making reverse turns and loops. Alpha
helices and beta strands are connected by these turns and loops. Most proteins have
compact, globular shape owing to reversals in the direction of their polypeptide
chains, which allows the polypeptide to create folds back onto itself. In many reverse
turns, the CO group of residue i of a polypeptide is hydrogen bonded to the NH group
of residue i+3. A turn helps to stabilize abrupt directional changes in the polypeptide
chain. Loops are more elaborate chain reversal structures that are rigid and well

defined. Loops and turns generally lie on the surfaces of proteins so they often
participate in interactions between proteins and other molecules. In a loop, there are
no regular structures as can be found in helices or beta strands.
Two hypotheses have been proposed for the role of turns in protein folding. In one
view, turns play a critical role in folding by bringing together interactions between
regular secondary structure elements. This view is supported by mutagenesis studies
indicating a critical role for particular residues in the turns of some proteins. Also,
nonnative isomers of X-Proline peptide bonds in turns can completely block the
conformational folding of some proteins. In the opposing view, turns play a passive
role in folding. This view is supported by the poor amino-acid conservation observed
in most turns. Also, non-native isomers of many X-Pro peptide bonds in turns have
little or no effect on folding.

Beta Hairpin Turns

A motif is when secondary structure elements combine in specific geometric
arrangements. Beta hairpin turns are one type of arrangement; they are one of the
simplest structures and then are found in globular proteins. Upon turning, the
antiparallel strand can bind effectively through hydrogen bonding between the
carbonyl carbon and the peptide backbone nitrogen. It has been shown that 70% of
beta-hairpins are less than seven residues long; the majority being 2 residues long.
There are two types of two-residue beta hairpin turns. The first, Type I, forms a lefthanded alpha-helical conformation. This left-handed conformation has a positive phi
angle due to the properties of the aforementioned amino acids. Glycine does not have
a side chain to sterically interfere with the turned amino acid sequence. Asparagine
and aspartate both readily form hydrogen bonds with the carbonyl oxygen as a
hydrogen bond acceptor. The second amino acid in the Type I turn is usually glycine
due to steric hindrance that would result using any amino acid with a side chain. In a
Type II beta hairpin turn, the first residue can only be glycine due to steric hindrance.
However, the second residue is usually polar, such as serine or threonine.

Fibrous proteins
Fibrous protein such as alpha-keratin and collagen consist of two right handed alpha
helix intertwined to form a type of left handed super-helix called an alpha coiled coil.
The two helices in this type of protein usually cross-linked by weak interaction such
as Van der Waals forces force and ionic interaction. The side chain interaction can be
repeat every seven residues, forming heptad repeats. Another form of fibrous protein,
that of collagen, exists as three helical polypeptide chains. These chains are relatively

long, ~1000 residues, and because of overcrowding, glycine appears once every three
residues. While the helix is stabilized by the steric repulsions, the three strands are
stabilized by hydrogen bonding. These protein usually serve structural roles in
organisms, alpha-keratin is commonly found in the cytoskeleton of a cell, as well as
certain muscle proteins. Collagen is often found in teeth, skin, and tendons.

Secondary Structure Prediction

The science of predicting what polypeptide chain will conform to which secondary
structure group (alpha-helix, beta-sheet/strand or turns/loops) is not particularly exact.
However, various frequencies of secondary structure formation of certain amino acids
have been recorded in actual scientific experimentation, and these values can allow
scientists to predict the folding of a protein based on its amino acid composition with
about 60-70% accuracy. Stretches of six or less residues can usually be predicted with
this accuracy. Although, certain amino acids tend to fold in its preferred conformation,
there are of course exceptions and so secondary structure prediction is not always
accurate. Tertiary interactions, interactions with residues further apart from each other,
can also determine the folding structures. Each amino acid has a preference for either
secondary structure, but it normally is only a small preference towards one in
comparison to another, therefore, this unfortunately does not mean much. Amino acids
can appear in an alpha-helix in one protein and also in a beta-sheet in another. Due to
the unpredictability of the secondary structure based on the sequence of amino acids,
secondary structures are being analyzed and predicted in relations to a similar family
of sequences.
Various techniques have risen throughout history in the study of secondary structural
prediction. With the aid of computers, prediction has been a pursued research topic in
bioinformatics and many approaches continue to be proposed. After Linus Pauling and
Robert Corey discovered the periodic alpha helix and beta sheet structures within
proteins in 1951, further elucidation of protein structure prediction began to grow. A
major method in secondary structure prediction was the Chou-Fasman[[1]] method; it
yielded a 50-60% accuracy. This method based its predictions on assigning a set of
prediction values to a certain amino acid residue and then applied an algorithm to that
value. Shortly after, further improvements were made on this method, the GOR
method[2], which was developed in the late 1970's and utilized entropy and
information concepts[3] for secondary structure prediction. When devised, the method
was about 65% accurate, however, improvements have also been made to it. There are
deductive techniques in which similar sequences are found in already identified
proteins. This method is accomplished by having computer software search databases
of identified proteins. Opposite of that would be the Ab initio method, which builds 3-

dimensional models without looking at similar residue sequences. This method is

based on hydrogen bonding principals and localization.
Other methods and factors of folding prediction include analyzing the basic chemical
tendencies of the side chains of amino acids to determine its preference in secondary
structure. The alpha-helix is taken as the default structure, thus amino acids that
destabilize alpha-helices are often found in beta-pleated sheets or loops and turns. For
instance, valine, threonine, and isoleucine will often destabilize the helix because of
branching of the beta carbon. These three amino acid residues are more often found in
beta-pleated sheets, where their side chains will lie in a separate plane than the main
chain. There are also amino acid residues that prefer neither alpha-helices nor betapleated sheets, for example, Proline has a restricted phi angle of ~60 degrees and no
NH group, all due to the fact that it is cyclic. This will disrupt both alpha-helices and
beta-pleated sheets, thus is found mostly in loops and turns. A counter-intuitive
example is glycine which, according to its small size, theoretically can fit in any
structure easily, but in reality it tends to avoid alpha-helices and beta-sheets also. The
folding definitely also relies on chemical interactions between the side chains so the
surrounding amino group interactions also affect the tendency of folding. These
tendencies are reflected in the frequencies of secondary structure for individual amino
acids.
The relative tendencies of secondary structures for particular amino acids are listed
below:
alpha-helix: Glu, Ala, Leu, Met, Lys, Arg, Gln, His
beta-sheet: Val, Ile, Tyr, Cys, Trp, Phe, Thr
turns and loops: Gly, Asn, Asp, Pro, Ser

Torsion Angles
Tosion angles are also called dihedral angles. The torsion angle is the measure in
degrees in bonds between atoms. Folding of proteins are influenced by the degree of
rotation amino bonds can hold. There are two different types of torsion angles existing
in polypeptide bonds. Phi, is the angle between the -carbon and the nitrogen atom
of a peptide bond. The other bond is called phi, which is the angle between the carbon and the carbonyl group. To measure , one must look from the nitrogen atom
towards the -carbon to measure if the angle is negative or positive. The angle is
negative if the -carbon rotates counterclockwise and vice versa. Furthermore, to

measure , one must look from the nitrogen atom towards the carbonyl group.
Likewise, the angle is negative if the carbonyl group rotates counterclockwise and
vice versa.

Ramachandran Diagram
The Ramachandran Diagram, created by Gopalasamudram Ramachandran, helps to
determine if amino acids will form alpha helices, beta strands, loops or turns. The
Ramachandran Diagram is separated into four quadrants, with angle as the x axis
and angle as the y axis. The combinations of torsion angles will put the amino acids
in specific quadrants, which determine whether is it will form an alpha helix, beta
strand, loop, or turn. Those that fall in quadrants 1 and 3 a few times in a row form
alpha helices, and those that repeat in quadrant 2 form beta strands. Quadrant 4 is
generally disfavored because of steric hinderance. Also, it is mostly impossible
because the different torsion angles combinations in quadrant 4 can't exist because
they cause collisions between the atoms of the amino acids. If the amino acids land in
the different quadrants, with no repeats, then they become loops or turns.
Furthermore, the principle of steric exclusion states that two atoms cannot occupy the
same place simultaneously.

Tertiary Structure
The tertiary structure of a protein is the three-dimensional structure of the protein.
This three-dimensional structure is mostly determined by the amino acid sequence,
which is denoted by the primary structure of the protein, however the amino acid
sequence cannot entirely predict on how the three-dimensional structure is formed.
Another contributing factor to the final shape of the tertiary structure is based on the

environment in which the protein is synthesized. The tertiary structure is stabilized by

the sequence of hydrophobic amino acid residues in the backbone of the protein.
Tertiary structure is formed by interactions between side chains of various amino
acids - in particular disulfide bonds formed between to cysteine groups. At this stage,
some proteins are complete, while other proteins incorporate multiple polypeptides
subunits which creates the quaternary structure.
Nucleation-condensation model- The tertiary folding process is very structured with
key intermediates. When a protein starts to fold, localized areas of the protein first
begin folding. Then, the individual localized folds come together to complete the
tertiary structure. The key concept is that when a correct fold is achieved, that fold is
retained until all other parts of the protein are also correctly folded. This folding
process follows reason because a random trial and error folding process would not
only take much more time to complete, but also would require much more input
energy.

Structure

A lobster's exoskeleton is an example of keratin.

A dog's fur is also an example of keratin.

Cysteine, an amino acid containing a thiol group, is responsible for the disulfide
bonds that hold a tertiary structure together. In the tertiary structure, when two helices
come together, they may be linked by these disulfide bonds. When there are few
disulfide bonds, less rigid substances will be formed and will lead to formation of
proteins such as hair and wool. These have a less rigid structure and are flexible, but
still are strong and resist breakage. Tertiary structures that contain more crossed
disulfide bonds, formed by cysteine residues, lead to stronger, stiffer and harder
structures such has exoskeleton. Others examples of proteins that contain more
disulfide bonds include claws, nails, and horns.
A structure made of two a-helices such as keratin can be found in living organisms.
Immunoglobulin, also known as antibodies, is an example of an all beta-sheet protein
fold. It consists of approximately 7 anti-parallel beta-strands arranged in 2 beta-sheets.
For instance, if a cysteine is mutated to another amino acid it can code to a different
protein which would lead to incorrect folding.

Domains
Some polypeptide chains fold into several compact regions. These regions in a
polypeptide chain are called domains and generally range from 30 to 400 amino acids.
On average, domains contain roughly 100 amino acids. Each domain forms its own
tertiary structure which contributes to the overall tertiary structure of the protein.
These domains are independently stable. Stabilization is caused by metal ions or
disulfide bridges that cause the folding of polypeptide chains. Different proteins may
have the same domains even if the overall tertiary structure is different.
There are four types of domains:

All- domains - Domains made purely from -helices.

All- domains - Domains made purely from -sheets.

+ domains - Domains made both of -helices and -sheets.

/ domains - Domains made from both -helices and -sheets layered in a

,, fashion with a -helix sandwiched in between 2 -sheets.

Mutations

In order for a protein to be functional (except in food), it must have an intact tertiary
structure. If a tertiary structure of a protein is disrupted, it is said to be denatured.
Once a protein is denatured, it will not be able to perform its intended or original
function. A primary cause for an alteration of the tertiary structure is a mutation in the
gene encoding a protein. The mutation in the gene can cause a domino effect that will
lead to the degradation of the tertiary structure. Degradation can cause several
diseases, one of which is called cystic fibrosis. Cystic fibrosis is brought about by a
mutation of a genes called cystic fibrosis transmembrane conductance regulator
(CFTR). This disease causes the exocrine glands to overproduce mucus. Most
commonly, CF patients suffer from lung failure by the age of early 20-30. Diabetes
insipidus, familial, hypercholesterolemia, and Osteogenesis imperfecta are also
diseases that originate from degraded proteins. A mutation in the tertiary structure
itself, rather than from a mutation in the nucleotide sequence can also lead to diseases.
Such mutated proteins can also aggregate and become insoluble deposits called
amyloids, and therefore lose the ability to function. A common mutation is when a
hydrophobic R group folds in, rather than out, in a hydrophobic environment. The
inherited form of Alzheimer's disease is one disease that is caused by mutated tertiary
structure. Another disease is "mad cow" disease, which is caused due to a-helix
(which are soluble) mutating into b-sheets (which are insoluble and cause amyloid
deposits). [4]

Folding
The folding of a protein is dependent on the amino acid sequence laid out in the
primary structure. It is also dependent on the environment in which the folding occurs.
In a hydrophobic environment, the hydrophobic side chains of the amino acids of the
protein fold out while the hydrophilic side chains fold in and vice versa for a
hydrophilic environment. An example of a protein that is folded in a hydrophobic
environment is Porin. Its hydrophilic side chains are folded in which creates a channel
for water to pass through. Amino acids that have nonpolar/hydrophobic side chains
such as leucine, valine, methionine, phenylalanine, and isoleucine would be folded out
in the folding of the protein in a hydrophobic environment. Likewise, in a hydrophilic
environment, amino acids with polar side chains such as glutamine and asparagine
fold outwards and the hydrophobic side chains would fold inwards.

Determination of Tertiary Structure

The tertiary structure of a protein is determined through X-Ray Crystallography and
Nuclear Magnetic Resonance (NMR) Spectroscopy. X-ray Crystallography was the
first method used to determine the structure of proteins. X-ray crystallography is one

of the best methods because the wavelength of an x-ray is similar to that of covalent
bonds found throughout proteins, creating a clearer visualization of a molecule's
structure. The scattering of x-rays by electrons is analyzed to determine the structure
of proteins. In order to use x-ray crystallography, the protein in question must be in
crystal form. Some proteins crystallize readily, while others do not. For those proteins
that do not crystallize readily, nuclear magnetic resonance (NMR) spectroscopy must
be used to determine its structure. NMR spectroscopy uses the spin of nuclei with a
magnetic dipole and chemical shifts to determine a molecules relative position.

Quaternary Structure

Atomic structure of the 50S Subunit from Haloarcula marismortui. Proteins are shown
in blue and the two RNA strands in orange and yellow.[11] This is an example of the
tertiary structure of the large unit of a ribosome
A quaternary structure refers to two or more polypeptide chains held together by
intermolecular interactions to form a multi-subunit complex. The interactions that
hold together these folded protein molecules include disulfide bridges, hydrogen
bonding, hydrogen bonding interactions, hydrophobic interactions interactions and
London forces. These forces are usually conveyed by the side chains of the peptides.
These polypeptide chains are the subunits of a protein, capable of taking part in a
variety of functions such as serving as enzymatic catalysts, providing structural
support in the cytoskeletons of cells, and even composing the hair on our heads.
The peptides of the protein can be identical or different. Insulin is a dimer consisting
of two identical peptides, while Hemoglobin is a tetramer consisting of two identical
alpha subunits and two identical beta subunits.

Naming Quaternary Structures

In naming quaternary structures, the number of subunits (tertiary structure) and the
suffix -mer (Greek for "part, subunit")are used:

1 subunit = Monomer
2 subunits = Dimer

3 subunits = Trimer

4 subunits = Tetramer

The pattern continues with pent-, hex-, hept-, oct-, and so forth.

Dimers

Computer-generated image of insulin hexamers highlighting the threefold symmetry,

the zinc ions holding it together, and the histidine residues involved in zinc binding.
Insulin
o Dimer alpha chain and beta chain
o

Linked by 2 disulfide bridges

HIV Protease
o

Dimer

Composed of identical subunits

Trimer

Collagen
o Composed of 3 helical polypeptide chains

Glycine appears at every third residue because there is no space in center

of the helix

Stabilized by steric repulsion of the pyrrolidine rings of the proline and

hydroxylproline residues

Hydrogen bonds hold together the strands of the collagen fibers

Tetramer

Structure of human hemoglobin. The protein's and subunits are in red and blue,
and the iron-containing heme groups in green. From PDB 1GZX Proteopedia
Hemoglobin
Hemoglobin
o Consists of 2 alpha and 2 beta groups

Has a globular shape

Has reverse turns that contribute to circular shape of the protein

Aquaporin
o

Made of 6 alpha helices

Form hydrophobic loops

Forms tetramers in the cell membrane with each monomer acting as

water channels

Breaking Apart the Quaternary Structure

The quaternary structure of a protein can be denatured by breaking the covalent and
non-covalent forces that keep it together. Heat, urea or guanidinium chloride will
denature a protein by disrupting the non-covalent forces, while betamercaptoethanol will break disulfide bridges by reducing the bridges.

[edit] Protein Folding

A protein is never "half folded", at the point where the concentration of the denaturant
is in between that of the folded and unfolded form of the protein, there are two
structures that exist. Folded and Unfolded, at a ratio of 1:1
Proteins are either folded, or not. There does not exist a stage where a protein is
"half-folded". This can be observed by slowly adding denaturant to a protein. This
will result in a sharp transition, from the folded state to the unfolded state,
suggesting there only exist these two forms. This is a result of cooperative transition.
For instance, if a protein is put in a denaturant where only one part of the protein is
unstable, the entire protein will unfold. This is due to the domino effect where
destabilizing one part of the protein will in turn destabilize the remainder of the
structure. When a protein is in conditions which correspond to the middle of the
transition between folded and unfolded, there is a 50/50 mixture of folded and
unfolded protein, instead of 'half-folded' protein.
After all is said about being in one structure or the other, there must be something in
between them on an atomic level. Unfortunately, this is an area that is still under
development, and much research is still being done. Theories such as the condensation
Nucleation Principle are concerned with this area of protein folding.

Analogy

If one takes each student in a class to be a different amino acid, each right hand to be
an alpha-carboxyl group, each left hand to be an alpha-amino group, and the head to
be the R group; then by joining right hands to left hands, the class will form a
polypeptide. The "bonds" joining the hands will be peptide bonds. This can be
considered the primary structure of a protein.
If one then takes students and "attract" them to other students 4 "bonds" away, this
structure will then fold into a secondary structure; namely the alpha-helix. If the
students were put into lines and were attracted to respective students in another line,
they would form a beta-pleated sheet.
Now imagine that the heads, or R groups, vary in areas such as personalities, or
polarity, like will attract like. The people who are more compatible will then gather
together, for instance, hydrophobic areas will usually gather together in the center
while surrounded by hydrophilic areas. This makes up the tertiary structure.
Now add in a different class, the people from the new class would have their own
tertiary structure, these new people will then come in and react with the original class
to form quaternary structures.

Human attempt to manipulate protein

assemblies (Quaternary Structures)
Controlling the quaternary structures is currently catching more and more interest in
academics. There are many advantages in manipulating protein assemblies. Firstly,
people are able to grow/synthesize enzymes that are beneficial to human. Yet, to get
these enzymes to work is the hard part. For example, nitrogenase, the enzyme that can
fix nitrogen gas to yield ammonia, can only work under aerobic environment and
coupled with ATP as energy source. In addition, researchers have revealed that
nitrogenase is compose of two proteins, one for ATP coupling&electron source and
the other is the reactive center for nitrogen fixation. The two protein assemble to work
as a whole. Recently, scientists remove the ATP coupling protein and replace it with a
Ruthenium complex. It turned out that Ruthenium complex can provide electrons with
light exposure. Now scientists don't have to deal with the complicate chemistry of
coupling ATP, but just shine lights on engineered nitrogenase to get it work! Secondly,
protein assemblies can have a lot of clinical/material applications. Ferritin is a family
of high-order protein assembly family, usually 12mers or 24mers. Previous researches
showed it can absorb large amount of Fe ion. Many researchers are working to control
the association and disassociation of Ferritins, seeking for solutions of drug delivery,

gas storage, metal harvest and etc. Many approaches have been developed to control
protein assembling. Following are some of them.

thumb|This is the complete structure of Nitrogenase. (from PDB)

1. Transition metal-directed. Metal centers in protein are important, not only because
they are reactive centers, but also they help stabilize the shape of protein by
coordination. Many amino acids are ligands by themselves. Cysteine, Histidine, lysine
are the common ones. Plus, researchers can engineer inorganic ligands onto proteins
by cysteine substitution. Thus, introducing inorganic ligands much broaden the
horizon of protein assemblies.

the structure of Phenanthroline (inorganic ligand).

the structure of Terpyridine (inorganic ligand).

Metal-ligand bonding has several properties. Most obviously, it is a strong interaction.
It is stronger than hydrogen bond and weaker than colvant bond. Therefore metalligand bond is strong yet not so strong that it is still reversible. Spatially speaking,
metals have its coordination orientation, mostly, octahedral and tetrahedral. This
property provides human great convenience in arranging proteins spatially.

shown is the cartoon model of a dimer of two terpyridine-labeled proteins.

shown is the cartoon model of a trimer of three phenanthroline-labeled proteins.

2. Hydrophobic interaction. In aqueous environment, amino acid with hydrophobic
side chains tend to aggregate together to minimize the exposure to water. Researchers
utilize this character and engineer certain matching pair of non-polar amino acids onto
proteins to obtain protein oligomers in water solution.
3. Salt bridges. It is well known that amino acids have different pI's. So at certain pH,
some amino acids are negatively charged, some are positively charged. If an area on a
protein is occupied by mostly negatively charged amino acid and another area is
occupied by positively charged amino acids, proteins can aggregate by electro static
attraction. However, this technique is usually not so selective.
More technique to direct protein assemblies are being investigated, such as coiledcoil. Human's ability to control quaternary structures is promising.