CHAPTER 4: PROTEINS: THREE-DIMENSIONAL STRUCTURE AND FUNCTION

Polypeptide Chains are Not Linear

Can contain all kinds of shapes
Conformation: a spatial arrangement of atoms that depends on the rotation of a bond or
bonds, can change without breaking any bonds
Configuration: can only be changed by breaking and re-forming covalent bonds
o Every protein has many potential configurations, but usually falls into its native
conformation
Each chain is encoded by a single gene
Proteomics: the study of large sets of proteins
Globular proteins: water-soluble, compact, spherical, tightly folded polypeptide chains,
hydrophobic interior, hydrophilic surface
Fibrous proteins: structural proteins that provide support to cells or organisms
o Are large cables or threads
Four Levels of Protein Structure
Primary structure: the linear sequence of amino acid residues in a protein
Secondary structure: regularities in local conformations maintained by hydrogen bonds
between amide hydrogens and carbonyl oxygens of the peptide backbone
o A helix, B sheet and turns
Tertiary structure: describes the completely folded and compacted polypeptide chain
o Many consist of several distinct globular units (domains) linked by a short stretch
of AA residues
o Stabilized by interactions of AA side chains in non neighboring regions
Quaternary structure: the association of 2 or more polypeptide chains into a multisubunit
protein
Methods for Determining Protein Structure
Primary structure discussed in Chapter 3
X-ray crystallography used for 3D structure
o Beam of X-rays shot at crystal of protein molecules and the electrons diffract the
rays and it is recorded on a film
o Electron density map shows overall shape of the molecule and the positions of
each atom, is used to determine the whole molecule
o Can be used without the AA sequence
Can be depicted in a space filling model , which shows each atom as a sphere, used to
show overall shape of protein
o Cartoon ribbon model, emphasized backbone of chain, doesnt show AA side
chains, but does show folding
o Other models combine the two in order to look at specific binding sites
Conformation of the Peptide Group
Peptide group consists of the 2 atoms in the peptide bond and the carbonyl O, the amide
H, and the 2 adjacent a-carbon atoms
The peptide bond b/w C and N is shorter than a normal C-N bond, but longer than a
normal C-N double bond

Bond b/w carbonyl C and O is longer than normal C-O double bond
Restricted to trans or cis conformations
o Trans: the 2 a-carbons of adjacent AA residues are on opposite sides of the
peptide bond and at opp corners of the rectangle formed by the peptide group
o Cis: the 2 a-carbons are on same side of peptide bond and are closer together
o Arise during synthesis and dont easily interconvert after peptide bond has formed
o Mostly all in trans conformation
o Free rotation restricted by steric interference of carbonyl Os and bulky side
chains
Ramachandran plots: charts the rotation angles of the Ca-C and the N-Ca bonds and
shows the sterically permitted values
The a Helix
Can be either a L or R handed screw, but almost always R
The rise(advance per AA residue up the vertical axis) is ideally 0.15nm
The pitch (advance per turn) is 0.54nm
Most have 3.5-3.7 residues per turn
Each carbonyl O in the backbone is hydrogen-bonded to the backbone amide H of the 4th
residue further towards the C-terminus
o Each hydrogen bond closes a loop of 13 atoms
o Stabilizes the structure
Phi and psi angles are similar
AA with large bulky side chains are found in a Helices less often
Many have hydrophobic AA on one side and hydrophilic on the other
B Strands and B Sheets
B strands: portions of polypeptide chain that are almost fully extended
o Each residue is about 0.32-0.34nm
o Proteins rarely contain isolated B strands
B sheets: when multiple B strands are put side by side
o Held together by H bonds b/w carbonyl oxygens and amide Hydrogens
o Can either be parallel, running in the same N- to C- terminal direction, or
antiparallel, running in opposite N- to C- terminal directions
Antiparallel sheets have almost perpendicular H bonds to chain
Parallel sheets less stable
o Can also be called a B pleated sheet
Side chains point alternately above and below sheet plane
A sheet can have 2-15 strands, each strand has avg. of 6 AA residues
Strands often twisted and sheet is distorted and buckled
Hydrophobic and hydrophilic side chains may separate by sides
Loops and Turns
Proteins can also contain stretches of non-repeating 3D structure (about 1/3 of residues)
o Phi and psi values are well within permitted range
Connect a helices and B strands
Allow chain to fold back on itself producing its 3D shape

Loops: contain hydrophilic residues, found on the surface of proteins, exposed to solvent
and form H bonds w/ water
Turn: a loop with up to 5 residues and cause an abrupt change in direction of the chain
(reverse turn)
o B turn: connect different antiparallel B strands
I and II, both contain 4 AA residues, stabilized by H bonds b/w carbonyl O
and amide H of the 4th residue
Produce about 180degree turn
Tertiary Structure of Proteins
Results from the protein folding
Stabilized by noncovalent interactions b/w the side chains of the residues
Supersecondary Structures
Also called motifs: recognizable combos of a helices, B strands, and loops that appear in
a number of different proteins
Can be associated with a particular function
Some common ones:
o Helix-loop-helix: occurs in calcium binding proteins, Ca binds to glutamate and
aspartate residues in the loop (can also be helix-turn-helix in DNA-binding
proteins)
o Coiled-coil: 2 amphipathic a helices interatcting thru hydrophobic edges
o Helix bundle: multiple helices with opposite orientations
o BaB unit: 2 parallel B strands w/ a helix in the middle connected by 2 loops
Connects to N-terminal of 1 B and the C-terminal of the other B
o Hairpin: adjacent antiparallel B strands connected by a turn
o B meander: antiparallel B sheet of sequential B strands connected by loops or
turns
o Greek key: 4 antiparallel B strands, 1 and 2 are in the middle
o B sandwich: B strands or sheets stack on top of each other
Domains: discrete, independently folded compact units
May consist of several motifs
Can range from 25-300 AA
Connected by loops and bound by weak interactions of side groups
Some domain structures occur in many proteins, others are unique
Proteins grouped into families according to similarities in domain and AA sequence
Can be classified into 4 categories: all-a, all-B, a/B if a and B alternate, and a+B if the
domain has separated clusters of helices and sheets
Can also be classified further by presence of folds
Domain Structure, Function, and Evolution
A single domain has a particular function
o Such as in multifunctional enzymes, each activity is associated with a single
domain
Intrinsically Disordered Proteins
Common and lack of secondary or tertiary structure encoded in AA

Many interact with other proteins

Contain short AA sequences within disordered region that act as binding sites

Quaternary Structure
Arrangement of subunits with their own multiple subunits, with each subunit being a
separate polypeptide chain
Oligomer: multisubunit protein
Dimers and tetramers predominate
Subunits have a defined stoichiometry and are held together by weak noncovalent
interaction
Determination:
o Molecular weight of native oligomer estimated by gel-filtration chromatography
o Molecular weight of each chain determined by SDS-polyarylamide gel
electrophoresis
Why proteins consist of multiple subunits:
o Oligomers are more stable than subunits, prolongs life of protein in vivo
o Active sites of some oligomeric enzymes formed by residues of adjacent
polypeptide chains
o Biological activity regulated by the changes in quaternary structure when they
bind ligands
o It is more efficient to have proteins that perform multiple tasks than to have whole
new proteins that duplicate part of the others function
o Channeling: product of one reaction can become the substrate of a second reaction
and both reactions can take place with the same enzyme because it has multiple
subunits
Most proteins have dyad symmetry: then can be split into 2 symmetrical parts
protein machine: a large complex of polypeptide components that carry out complex
reaction
Protein-Protein Interactions
Many kinds of forces that hold subunits together
Interaction can occur in a small patch or over a large portion of the polypeptides
Other interactions include contact b/w proteins and receptors and those b/w various
enzymes
P1 + P2 P1:P2
Ka = [P1:P2]
[P1][P2]
Subunit interactions are very strong, protein-protein interactions are extremely weak
As conc of P1 and P2 increase, it is more likely they will bind to each other
At some point, rate of binding = rate of dissociation
Protein Denaturation and Renaturation
Denaturation: environmental change or chemical treatment that disrupts native
conformation and causes loss of biological activity
o Usually only a small amount of energy required, usually by heat

o Broken bonds, unfolding and loss of structure

o Takes place over a small temp range, breakage of only a few bonds required for
complete loss of conformation
o Chemical denaturation: chaotropic agents and detergents allow water molecules to
solvate nonpolar groups in the interior of a protein, disrupting the hydrophobic
interactions that hold the protein together
o If a protein contains disulfide bonds, they need to be cleaved (reduced) as well for
denaturation to occur
Renaturation can take place, but can go wrong, disulfide bonds dont always reconnect
correctly, but PDI enzymes can correct these bonds
Protein Folding and Stability
Proteins fold into their correct shape as it emerges from the ribosome
Naturally folds into a low-energy conformation
Cooperative effects occur: the first few interactions trigger the next ones, adopting lower
and lower energies as it goes
Very rapid
Different domains fold independently
No folding pathway has ever been described, scientists mainly focus on intermediates
Forces that Stabilize Protein Structure:
Hydrophobic Effect
Nonpolar side chains are hydrophobic, they associate with themselves and collapse to the
interior and hydrogen bond with each other, neutralizing their polarity
Polar side chains remain in contact with water
Hydrogen Bonding
H bonds in the a helices and B sheets and turns form first, then b/w polypeptide backbone
and water, backbone and side chains, and side chains and water
Van der Waals Interactions and Charge-Charge Interactions
Van der Waals b/w nonpolar side chains
Charge-charge interaction b/w oppositely charged side chains
Folding Assisted by Molecular Chaperones
Folding doesnt involve a random search for the native conformation and depends largely
on primary structure
Molecular chaperones increase rate of correct folding by binding new polypeptides
before they are completely folded, which prevents incorrect folding
Collagen
Major protein component of connective tissue, 30% of total protein in mammals
Various forms and functions
3 L-handed helices coiled up to form a supercoil
Each helix is more extended than a normal a-helix, pitch and rise are larger
Triple helix stabilized by hydrogen bonds
o Form between a glycine residue of 1 chain and a carbonyl O in another chain
o 1 H bond for every 3 residues

Contains an amino acid called hydroxylysine which sometimes bond to carbs, which
makes collagen a glycoprotein
Myoglobin and Hemoglobin Structure
Myoglobin facilitates diffusion of oxygen to muscle tissues of birds, reptiles, and
mammals
o Part of globin family
o Bundle of 8 a-helices
o Interior made up of highly hydrophobic AA residues, stabilizes protein
o Heme prosthetic group occupies space created by 3 a-helices and 2 loops
Hemoglobin: tetrametric protein that carries oxygen in blood
o 2 different globin subunits a- and Bo Contains 2 a-chains and 2 B-chains
o Each subunit is similar to myoglobin and contains a heme prosthetic group
o The a- and B- subunits interact and are slightly different
Red color due to the heme group (Iron surrounded by 4 N, which part of 5 member rings,
each ring is connected by a methyl group)
o Iron has a 2+ charges
Oxygen Binding to Myoglobin and Hemoglobin
Oxygen Binds Reversibly to Heme
Oxygenation: reversible binding to the heme group
o Deoxy- and oxy-myoglobin/hemoglobin
Heme group is partially buried in the molecule
The 4 nitrogens and 2 histidine residues surround iron in a tetrahedron (oxymyoglobin)
Deoxymyoglobin only has 5 ligands
Side chains block entrance to iron, so protein vibrates rapidly to allow entry
An electron is partially transferred to the O2 molecule, the crevice made by the side
chains prevents a complete bond from forming
Oxygen Binding Curves
Plots fractional saturation against concentration of O2
o Fractional saturation is ratio of saturated molecules over total molecules
Hemoglobin is an Allosteric Protein
Binding and release of oxygen regulated by allosteric interactions
o Small specific molecule (allosteric moderator) binds to a protein and controls
its activity
When O2 is transported to metabolizing tissues, pH is lower so oxygen is released
and CO2 binds to hemoglobin (reverse happens in lungs)
o Usually transported at HCO3
Antibodies Bind Specific Antigens
Antibodies are a specific protein that recognize and bind antigens
o Many different foreign compounds act as antigens that produce an immune
response
Antibodies synthesized by lymphocytes

Animals produce a huge array of different antibodies that stay low for years until needed
again
When antigen binds to lymphocyte, it releases antibodies into bloodstream that bind the
antigen and mark it for destruction by the lymphocyte
Most abundant antibodies are IgG, which are Y-shaped
o 2 light chains and 2 dark chains bound by disulfide bonds and have 2 carbs bound
to the heavy chains
o Ends of the top of the Y have 2 NH3+ which bind to the antigens