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 chargecharge 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.

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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

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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

Chaperonins

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 righthanded 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

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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 2,3bisphosphoglycerate (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

O2 Binding induces conformation change

T-conformation

R-conformation

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 Tconformation (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 alphaamino group to form carbamate group. --N3H+ + HCO3-  -NHCOOCarbamation 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.