Protein Folding|Graduate Biochemistry 5| Tulane

Protein Stability and Folding

Overview

• Post translational modifications
• The interactions that drive protein folding
• The interactions that drive protein unfolding
• The net stability of folded proteins
• Free energies of protein folding
• Mutation studies
• The Levinthal paradox
• Folding pathways
• Molecular Chaperones
• Degradation of proteins
  Reading: All of Chapter 6

Proteolysis

• segments-cleave
• signal peptide cleaved in Membrane/secreted proteins
• proproteins: hormones; enzyems
• The final hormone is cleaved

• Some proteases are synthesized as zymogens which are activated by cleavage of a segment from the protein

Significant: Table (half life), soluable
Exp: Insulin

PS: Some maturation of proteins are involved with pro-peptides cleavage. Insulin is an classical example.
Insulin has not much of structure amino acid, lack of an hydrophobic core.
6 Cystines and form 3 pair of disulfide bonds. Insulin’s primary structure has not enough information to drive it to the native structure. As a result, people believe that insulin has a memory of each processes to fold.

© PDB ID=2hiu

Amino Acid Modulation

Modulation Type	Added Group	Location / targets	Reversible / Not	General Features
Glycosylation	carbohydrates	-N-linkage (on N-R) (More common) -O-linked (on S, T)		Variety; long; branched; Charged; Ionized; Large
Hydroxylation	Carboxyl	L & P are hydroxylated on —CH₂ groups Requires Ascorbate (VC) mainly in collagens(Gly-X-Y) P - 3 or 4 hydroxy proline	Irreversible	Lack of VC, cell depart
Methylation	Methyl	K and H are methylated on –NH₃, NH₂ groups; K can also be trimethylated (3 Methyl groups)	Reversible	Doesn’t loose it’s charge. the Amine group still keep charge. Spatial Increased and effect the interactions with other atoms. DNA binding proteins (K phosphate the DNA)
Acetylation/Acylation	R-Carboxyl (myristate)	Amino group of the R chain to for a peptide bond K; H; Terminal AA	R Chain are reversible; Terminal are usually not reversible	Charge is removed. More hydrophobic Increase the interact with cell membrane. Reversible; Highly Controlled
Carboxylation	Carboxyl	The γ-carbon of E can be carboxylated		The E have two carboxyl groups. Mainly blood coagulation enzymes. Requires VK
Sulfation	Sulfate	the hydroxyl of T	Permenent	more polar, give hydrogen bonds large polar ionic sulfate group
Prenylation	Isoprenyl chains	C-terminal of C residue through a thioether (C-S-C) linkage.		Promoting interact with membranes Signal transduction proteins are prenylated
Amidation	Amnie group	C-terminal carboxyl group		polar but not ionizable.
Phosphorylation	Phosphate groups	The hydroxyl group in the R chain. T; S; Y H Could be also be phosphorylated in some case	Reversible	Large polar group

© Raimund Nagel, et al	© Wiki

Protein Folding

Interactions

The forces that stabilize folded proteins

• The hydrophobic effect
• Hydrogen bonding
• Ionic interactions
• van der Waals interactions (London dispersion force)
• Disulfide crosslinks
  cite: Kauzmann, W. (1959) “Some factors in the interpretation of protein unfolding” Adv. In Proteins Chemistry. 14:1.

The hydrophobic effect

• The unfavorable interaction between hydrophobic sidechains and water is reduced when a protein folds into its native structure
• Up to 75% of hydrophobic residues are buried in folded proteins
• Its contribution greatly favors folded state

Hydrogen bonding

• Every residue in a protein has at least one H-bond donor and at least one H-bond acceptor (the peptide bond)
• Concentration of H-bonding groups in a protein is ~25M
• Most potential H-bonding groups are, in fact, H-bonded in the interior of folded proteins
• H-bonds define secondary structure geometry

The forces that stabilize folded protein

• The hydrophobic effect
• Hydrogen bonding
• Ionic interactions
• van der Waals interactions (London dispersion force)
• Disulfide crosslinks

Ionic interactions

• Salt bridges form between acidic and basic residues
• Majority of salt bridges are on surface
• Buried salt bridges are conserved

van der Waals Interaction

• Contribution is from tight packing of protein core
• Protein density is ~1.3 g/ml (H20=1.0 g/ml Ethanol=0.8 g/ml)

Disulfide Crosslinking

• Covalent crosslinks between the sulfurs of cysteine residues
• Generally nonlocal
• Significant (but not dominant) contribution to stability

The forces that destabilize folded protein

• Conformational entropy
• Hydrogen bonds to water
• Electrostatic repulsion

Conformational Entropy

• Backbone of folded protein has only one (or a few) conformations.
• The backbone of an unfolded protein has 32n conformations (because there are 3 low energy φ and ψ torsion angles for each C_α)
  The backbone entropy change favoring unfolding:
  ΔS = Rln(3²ⁿ/1) = 2nRln(3) = 1.4 kcal/mol/residue (5.4 kJ/mol/residue)

Hydrogen bonds to water

• Every amino acid has at least two hydrogen-bonding groups
• The concentration of bulk water is ~55 Molar and each water molecule has 4 hydrogen bonding groups. Therefore the favorable energetic contribution of hydrogen bonds to folding may be small
  However, the unfavorable cost of unmade hydrogen bonds in the interior of the protein is still high

Hydrophobic amino acids are buried

Residue	Exposed	Burued	Interm ediate
S	0.70	0.20	0.10
T	0.71	0.16	0.13
A	0.48	0.35	0.17
G	0.51	0.36	0.13
P	0.78	0.13	0.09
C	0.32	0.54	0.14
D	0.81	0.09	0.10
E	0.93	0.04	0.03
Q	0.81	0.10	0.09
N	0.82	0.10	0.08
L	0.41	0.49	0.10
I	0.39	0.47	0.14
V	0.40	0.50	0.10
M	0.44	0.20	0.36
F	0.42	0.42	0.16
Y	0.67	0.20	0.13
W	0.49	0.44	0.07
K	0.93	0.02	0.05
R	0.84	0.05	0.11
H	0.66	0.19	0.15

C: Buried Residues
V; L; I; F; W; C

0.01: Exposed Residues
D; E; N; Q; K; R

Protein Unfolding

• An unfolded protein has lost its tertiary/quaternary structure and its biological activity
• Depending on the experimental conditions an unfolded protein may be stable or it may aggregate/precipitate

Treatments that unfold proteins:
High temperature: many proteins unfold at 50-90 º C
pH: some proteins will unfold when they gain a large net charge
Denaturants: proteins unfold in detergents like sodium dodecyl sulfate (SDS) or in denaturants like urea and guanidine
Unfolding curves are sigmoidal

© Agnishwar Girigoswami

Net Stability

• Most proteins have a net stability of ~10 kcal/mol/protein
  Compare this to the individual contributions:
• Hydrophobic effect ~ 1-2 kcal/mol/amino acid residue
• Conformational Entropy ~1-2 kcal/mol/amino acid residue
  For a 200 residue protein these contributions can be 400 kcal/mol each but the overall stability will always be ~10 kcal/mol

Mutational Studies of Protein Stability

© Lydia Chang

• Most mutations are destabilizing
  • The ability to precisely predict the change in folding energy (ΔG) is not very good
• Buried residues have a bigger effect than exposed residues

[图] Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol. 2002 Jul 5;320(2):369-87.

The Levinthal Paradox

Does a protein find its native configuration by a random search?
Carbon-carbon bonds reorient each ~ 10-13 sec
For a protein of n residues there are 32n
conformations
If n = 100, then 3²ⁿ ~ 10⁹⁵
If 10¹³ conformations were sampled every second it would take 10⁸² seconds to sample them all…
…but the age of the universe is only about 10¹⁷ sec!
It is now known that proteins fold on time scales from milliseconds to minutes

• Proteins fold via conserved, hierarchical pathways
  (This may explain the prevalence of the Greek Key motif)

PS:
why 3²ⁿ:

• 2: there are two tortion angles: φ and ψ
• n: n peptides
• 3: If each of these bond angles can be in one of three stable conformations^[1]

Diseases of protein misfolding

Cystic Fibrosis: Alzheimer’s disease
Mad Cow Disease: Retinitus pigmentosa

Molecular Chaperones

© Alexandra Richardson, 1998

“Heat shock proteins”
Molecular Chaperones
Chaperonins

Chaperones assist protein folding in the cell

• They bind unfolded chains
• They use energy from ATP to bind and release protein chains in a repeating cycle
• ATP turnover rate is slow 1 sec^-1
  The GroEL/GroES chaperone form E. Coli is well understood

Other Proteins

Protein Disulfide Isomerase
Peptidyl proline cis-trans isomerase

Protein Degradation in the Cell

There are two main pathways for protein degradation in eukaryotic cells
Lysosomal Degradation

• The lysosome is a membrane-encapsulated organelle
• It contains ~50 hydrolytic enzymes
• Lysosomes have acidic pH
• Has both selective and non-selective degradation processes
  Proteasomal Degradation
• The proteasome is a large protein machine (MW = 2x106)
• It degrades proteins that have the small protein ubiquitin attached to them
• Ubiquitination rate (degradation rate) is determined by the N-terminal residue
• Ubiquitin (76 residues) is the most highly conserved protein known

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1. Wikipedia: Levinthal’s paradox ↩︎