Lecture 6

Protein folding, 3-D Structure /

Function
 Protein folding and stability
• Protein folding does not involve a random search for the proper conformation.
• The final shape of a protein is dependent upon its primary structure.
• Small proteins can fold properly in vitro, but larger ones need the help of molecular
chaperones.
• As the protein folds, initial interactions then initiate further interactions –called the
cooperativity of folding.
• Folding occurs in less than a second.
• Protein folding and stabilization depend upon noncovalent forces, including the
hydrophobic effect, hydrogen bonding, van der Waals interactions, and charge-
charge interactions.
• Although individually weak, collectively they are strong.
• The weakness gives the protein flexibility to change conformations. The collective
effect keeps the protein in its proper shape.
• No actual protein folding pathway is known, however, the structure of some
intermediates has been described.
 Protein Folding
• Ribonuclease A (RNase A) will refold to native
structure spontaneously (1 minute)
• >1050 possible conformations
• If 10-13 sec per conformation would take 1030
years to sample enough to determine structure
• How do proteins fold so quickly?
 Protein Folding
• Structures of globular proteins are not static
• Proteins “breathing” between different conformations
• Proteins fold towards lowest energy conformation
• Multiple paths to lowest energy form
• All folding paths funnel towards lowest energy form
• Local low energy minimum can slow progress towards
lowest energy form
 Pathway of
Protein Folding

1) protein “collapses” onto itself

as result of hydrophobic effect

2)some parts of secondary structure

begin to form molten globule
3) Formation of domains
through aggregation of local 2o
structures

4) Adjustment of domains to
form native multidomain
protein
 Non-covelant bonds
The Hydrophobic Effect
Proteins are more stable when their hydrophobic R-groups are in the interior of a
protein and away from water. Nonpolar side chains then interact with each other.
Polar side chains remain in contact with water on the protein surface.
Hydrogen Bonding
• Hydrogen bonds in a helices, b sheets and turns form first as a protein folds.
• Many hydrogen bonds ultimately form between polypeptide backbone and water,
between backbone and R-groups, between R-groups, and between R-groups and
water.
• Those hydrogen bonds within interior of protein are more stable than those on the
surface because these bonds do not then compete with water molecules.
Van der Waals Interactions and Charge-Charge Interactions
• Van der Waals contacts between nonpolar side chains are also important.
• Charge-charge interactions contribute minimally to protein stability because most
ionic bonds are found on the surface of a protein.
 Disulfides Bonds
• Stabilize native structure
• Formed after native conformation achieved
• Abundant in secreted proteins but not in intracellular
proteins
• Protein disulfide isomerase catalyzes reduction of incorrect
disulfide linkages
 Chaperonins
• Protein complexes that promote protein folding
• Most chaperones are heat-shock proteins. The major heat-shock
protein is HSP-70
• Chaperonins don’t determine native structure
• Prevent misfolding and aggregation of protein
• Require ATP for protein binding, after ATP hydrolysis native protein
released
• bind to the hydrophobic portions of a protein and prevent them from
interacting with water
 Collagen a Fibrous protein
• Major component of connective tissue of vertebrates.25-35% of total protein in
mammals.
• Consists of three left-handed helical chains coiled around each other in a right-
handed supercoil.
• Each helix has 3 amino acids per turn and a pitch of 0.94 nm (0.31 nm/residue) 
more extended than an α-helix (0.54 nm, 0.15 nm /residue).
• Helical regions consist of the amino acids –Gly-X-Y, where X is usually proline
and Y is usually hydroxyproline.
• Pro, hydroxy pro prevent formation of α-helix and make collagen somewhat
rigid.
• Gly allow collagen to form tightly wound left that accommodates the proline
residue.
• Stability of the collagen helix is achieved via hydrogen bond forms between the
amide hydrogen atom of glycine residue in one chain and the carbonyl oxygen of
X residue in an adjacent chain.
• There are no intrachain hydrogen bonds.
• Hydroxyproline and hydroxylysine are made from proline and lysine after the
protein has been synthesized, the hydroxylation is catalysed by enzyme and require
Collagen super colloid
Covalent cross bond (Schiff base)
 Myoglobin/
Hemoglobin
• First protein structures
determined
• Oxygen carriers
• Hemoglobin transport O2
from lungs to tissues
• Myoglobin O2 storage
protein
Mb and Hb subunits structurally similar
•8 alpha-helices
•Contain heme group

•Mb monomeric protein

•Hb heterotetramer (a2b2)

myoglobin
hemoglobin
 Heme group
• prosthetic group
• Heme group in hydrophobic crevice of globin
protein
• Heme = Fe++ bound to tertapyrrole ring
(protoporphyrin)
• Iron atom in center can form 6 bonds: 4 with
nitrogens from protoporphyrin and 2 on either
side of plane.
• Iron atom can be in ferrous (+2) or ferric (+3)
state
• Heme non-covalently bound to globin proteins
through His residue
• O2 binds non-covalently to heme Fe++, stabilized
through H-bonding with another His residue
 Myoglobin structure
1) Extremely compact
2) 75% of structure in a-helix (8 helices, named A, B, C, ...H).
3) 4 of helices are terminated by proline residue
4) Main-chain peptide groups are planar
5) Little empty space inside molecule; interior consists almost
entirely of nonpolar residues; amino acids that are
amphipathic oriented so that hydrophilic portions face
exterior; only polar amino acids in interior are 2 histidines,
which are part of binding site.
 Oxygen binding site
• Heme group located in crevice in myoglobin molecule.
• Iron atom is bonded to histidine in F8 (histidine); the
oxygen-binding site on iron is located on other side of heme
plane (E7).
• Binding of oxygen to heme must occur in a bent, end-on
orientation.
• If only a small portion of the protein binds oxygen, why have
the rest of the protein?
• Heme exposed to oxygen by itself rapidly oxidizes to +3,
which cannot bind oxygen.
• Heme is much less susceptible to oxidation because not only
allows heme to bind oxygen, but it is a reversible process.
 CO binding
• Carbon monoxide is a poison because it combines with
ferromyoglobin and ferrohemoglobin to block oxygen
transport.
• CO’s binding affinity is about 200x stronger than that for
oxygen.
• If allow CO to interact with isolated iron porphyrins, the
iron, carbon, and oxygen atoms are in a linear array.
• If allow CO to interact with myoglobin or hemoglobin, CO
axis is bent, as in oxygen binding because of steric
hinderance from His E7 --> greatly weakens the interaction
of CO with the heme.
 Hemoglobin structure
• Hemoglobin consists of 4 polypeptide chains, 2 of one type, 2 of
another (α2, β2), held together by noncovalent bonds.
• Each polypeptide contains a heme group and oxygen binding
site.
• The three-dimensional structures of myoglobin and a and b
chains of hemoglobins are very similar --> myoglobin resembles
a chains of hemoglobin.
• Odd because amino acid sequence is not very similar -->
different amino acid sequences can specify similar 3-D
structures.
• Those amino acids found to be invariant (do not change) are
those directly bonded to heme iron or hold helices together.
• The nonpolar character of interior of molecule is conserved -->
important in binding heme group and stabilizing 3-D structure of
 Binding of O2 to Hb
Hemoglobin is more intricate than myoglobin.
1) transports protons, carbon dioxide, and oxygen
2) is an allosteric protein
3) binding of oxygen to hemoglobin is cooperative
4) affinity of hemoglobin for oxygen is pH dependent;
same true for CO2
5) hemoglobin also regulated by 2,3-
bisphosphoglycerate (BPG)
 Oxygen Binding Curves
•Mb has hyberbolic O2
binding curve
•Mb binds O2 tightly.
Releases at very low pO2
•Hb has sigmoidal O2
binding curve
•Hb high affinity for O2 at
high pO2 (lungs)
•Hb low affinity for O2 at
low pO2 (tissues)
Oxygen Binding Curve
Oxygen Binding Curve
 O2 Binding to Hb shows positive
cooperativity
• Hb binds four O2 molecules
• O2 affinity increases as each O2 molecule binds
• Increased affinity due to conformation change
• Deoxygenated form = T (tense) form = low affinity
• Oxygenated form = R (relaxed) form = high affinity
O2 Binding to Hb shows positive
cooperativity
 T-conformation R-conformation
O2 Binding
induces
conformation
change

Heme moves 0.34 nm

Exposing crystal of
deoxy-form to air cause
crystal to crack
 Allosteric Interactions
• Allosteric interaction occur when specific molecules
bind a protein and modulates activity
• Allosteric modulators or allosteric effectors
• Bind reversibly to site separate from functional binding
or active site
• Modulation of activity occurs through change in
protein conformation
• 2,3 bisphosphoglycerate (BPG), CO2 and protons are
allosteric effectors of Hb binding of O2
 Bohr Effect
• Increased CO2 leads to decreased pH
CO2 + H2O <-> HCO3- + H+
• At decreased pH several key AA’s
protonated, causes Hb to take on T-
conformation (low affinty)
• In R-form same AA’s deprotonated, form
charge charge interactions with positive
groups, stabilize R-conformation (High
affinity)
• HCO3- combines with N-terminal alpha-
amino group to form carbamate group.
--N3H+ + HCO3-  --
NHCOO-
• Carbamation stabilizes T-conformation
 Bisphosphoglycerate (BPG)
• BPG involved acclimation to
high altitude
• Binding of BPG to Hb causes
low O2 affinity
• BPG binds in the cavity
between beta-Hb subunits
• Stabilizes T-conformation
• Feta Hb (a2g2) has low
affinity for BPG, allows fetus
to compete for O2 with
mother’s Hb (a2b2) in
placenta.