Protein Folding

BIBC 100

The Folding Problem

• How Proteins Fold?– Consider a protein with 100 a.a.

• 10100 possible conformations (avg. of 10 conformations/a.a.)

• If it converts from one conformation to another in ~10-13 sec then the avg. time to sample all conformations would be 1077 years or 1085

seconds

Cosmic Term: longer than the life of earth/universe

Levinthal’s Paradox

• However, in vivo, proteins fold in 10-1-103 seconds, a mismatch of >98 orders of magnitude

• Conclusion: Folding is not random deterministic (directed)

• Native State (folded state)– Unique (action)– Stable (energy)– Accessible (kinetics)

U.S.A.

Second Genetic Code:

Sequence StructureFolding

Design(reverse folding)

Computer Algorithm

Input Output

Folding Sequence Structure

Design Structure Sequence

Importance?

1. Too many sequences and still few structures(105106, genome) (~105)

Understanding seq.-struct. relation requires solving:

2. Biotechnology –unleashed power Design: drugs, hormones, sensors, processes

(photosynthesis), imagine…

Too many structures or The Folding Problem

Characteristics of Folded State

• Tight packing – compact• Sequence determined/environment

modulated– (N-P) Search Space

• Families and symmetry• Each sequence unique structure• Native state is thermodynamically stable

(lowest energy)

USA Dogma

Protein Stability and Folding• A protein’s function depends on its 3D-structure

• Loss of structural integrity with accompanying loss of activity is called denaturation

• Proteins can be denatured by:

• heat or cold

• pH extremes

• organic solvents

• chaotropic agents: urea and guanidinium hydrochloride

• Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds

• Urea in the presence of 2‐mercaptoethanol fully denatures ribonuclease

• When urea and 2‐mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed   

• The sequence alone determines the native conformation

• Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize

Ribonuclease Refolding Experiment

Physics of Folding

• Enthalpy drives towards this– HB interactions– H bonding– Ionic interactions– Heat content of a system

Free Energy is the Difference– Folded state is more stable

• Entropy drives towards this– HB exposed– Disorder in a system

Steps of Folding

Unfolded bury core 2o Molten globule 3o 4o

protein HB aa (loose 3o)(breathing)

< ms Up to 1s

Energy Funnel for Folding

• Multiple folding pathways can occur

• Model this with energy funnel

N denotes the native fold and is the lowest free-energy state

H2AH2

Slide 20

H2 insert figure 4-29a and bHeather, 6/28/2012

AH2 4-29 c and d included--crop?Hug, Alyssa-Rae, 10/26/2012

Physical Forces

• H bond (local, near neighbors)• Hydrophobic (compactness/molton

globule, distant neighbor)

Folding Pathways FunnelsExplore the energy landscape or conformational space (degrees of freedom)

Proc. Natl. Acad. Sci. USA 89:8721-8725 (1992)

Protein folding follows a distinct path

ProteostasisMaintenance of cellular protein activity is accomplished by the

coordination of many different pathways

Protein misfolding is the basis of numerous human diseases

Computational Modeling• Major area of research• Infancy

– We still cannot accurately fold proteins by computer

Needed:1. Understanding process2. Defining the minimum3. Faster computers4. Models testable by experimentationThat’s why folding and design are two different

formulations of the same problem

In Vivo Folding

• Chaperones – bind to incompletely folded polypeptides– Prevent aggregation– Regulate translocation

• Foldases – catalyze foldingN I U

chaperonesNative Intermediate Folding

Rx’s: -Disulfide Bonds-x-pro peptide bonds-cis-trans isomers

Goal: To prevent aggregation (collapsed intermediates) and alternatively folded states

Why won’t it fold?

Most common obstacles to a native fold:

• Aggregation

• Non-native disulfide bridge formation

• Isomerization of proline

Chaperones prevent misfolding 

Chaperonins / Heat Shock Proteins HSPs help proteins fold by preventing aggregation

• Recognize only unfolded proteins– Not specific– Recognizes exposed HB patches– Prevent aggregation of unfolded or misfolded proteins

• HSP70– Assembly & disassembly of oligomers– Regulate translocation to ER

• HSP60 (GroEL) & HSP10 (GroES)– Work as a complex

• Each subunit– Apical ( motif)

• Opening of chaperone to unfolded protein

• Flexible• HB

– Intermediate ( helices)• Allow ATP and ADP diffusion• Flexible hinges

– Equatorial ( helices)• ATP binding site• Stabilizes double ring structure

– Central cavity up to 90Å diam.

• 7 subunits in one ring• 2 rings back to back

GroEL

• Cap to the GroEL

• Each subunit– sheet– hairpin (roof)– Mobile loop (int w/ GroEL)

• 7 subunits in functional molecule

GroES

GroEL+ GroES work together

• GroEL makes up a cylinder– Each side has 7 identical subunits– Each side can accommodate one unfolded

protein

• 1 GroES binds to one side of GroEL at a time– Allosteric inhibition at other site

• One side of cylinder is actively folding protein at a time

1. GroEL/ATP complex at side A2. Bind GroES on this side

7 ATP7 ADP this side has a wider cavity but closed topother side has smaller cavity and open top

3. Side B ring binds unfolded proteinGroES falls off of side AADP falls off of side A

4. Side B ring binds 7 ATPs5. GroES binds GroEL/ATP

7 ATP7 ADP protein folding occurs

6. Side A ring binds 7 ATPsprotein folding occurs7 ATP7 ADP (side A)7 ADP & GroES (side B) falls off

7. Side A ring binds next unfolded protein

Chaperonins facilitate folding

The two chambers alternate in binding and folding of client proteins

• Switch side of ATP binding each time• Switch side of GroES binding for each folding rxn• Switch side of protein docking for each folding rxn

Fink, Chaperone Mediated Folding, Physiological Reviews, 1999

Mechanism of Chaperonin Function

GroEL-GroES trapped encapsulating a folding intermediateCell 153, 1354–1365, June 6, 2013

Existence of Folding intermediates detected by NMR

• Obtained by analysis of the disulfide bonding pattern of intermediates trapped during reoxidation of a 59 a.a. protein (bovine pancreatic trypsin inhibitor)

• Barnase folding pathway-Fig. 6.4 & Kinemage• Role: transient structures in nascent chains –

could initiate early steps in folding (funnels)• Biotechnology – problematic inclusion

bodies

Sequence affects helix stability• Not all polypeptide sequences adopt -helical

structures

• Small hydrophobic residues such as Ala and Leu are strong helix formers

• Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible

• Gly acts as a helix breaker because the tiny R-group supports other conformations

• Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect formation

The Helix Dipole• Recall that the peptide bond has a strong dipole

moment– Carbonyl O negative– Amide H positive

• All peptide bonds in the helix have a similar orientation

• The helix has a large macroscopic dipole moment• Negatively charged residues often occur near the

positive end of the helix dipole

Protein Stability (thermal)• Protein engineering (mutagenesis)1. S-S bridges

a. -CH2-S-S-CH2-b. Analysis of all possibilities (many)c. Energy minimization to reduce to a few plausible candidatesd. Site-selective mutationse. Protein synthesisf. Assay:

example – T4 lysozyme (x-ray structure known)Reducing degrees of freedom (entropy) increases protein

stability

Protein Stability Cont…

2. Gly and Pro-Gly freedom-Pro Constraints (side chain is fixed by covalent bond to main chain- Gly Pro has propensity to increase stability (more delicate)- GlyAla usually increase- ProAla usually decrease

Protein Stability Cont…

3. Dipolar stability

N-end (-a.a.)C-end (+a.a.)

increase stability by mutating residues at N-end of helices from polar to negative (e.g. ASNASP, SERASP)

Helix:

Protein Stability

4. Hydrophobicity in the core (cavity)-Barnase (bacterial RNAse-110 a.a.)

-structure by both x-ray and NMR-introducing cavities in the core by mutations

such as IleVal or PheLeuCavity for a CH2

Stability by 1kcal/mol

-More delicate design-Needs structure

Prediction of Structure From Sequence

• Empirical – in progress• ~70% successful-at best (62-65%)• Essence: Pattern Recognition• Key: Evolutionary Information

– Sequence homology implies similarity in structure and function– By inference/By Anaysis

• Data bases (2007 >500,000 seq., 2013 >87,000 Structures Information

Prediction• Example: Homologous proteins

Conserved Core Variable Loop

A prokaryotic Kv channel with essential sequence conservation of the voltage sensor.

Santos J S et al. J Gen Physiol 2006;128:283-292The Rockefeller University Press

Secondary Structure Prediction for 3-Model

• Predict: α, β, loop, β-turn• Predict: membrane-spanning α-helix• Predict: Amphipatic structures

α β• Prediction of the folded structure of

tryptophan synthetase, and• the catalytic subunit of c-AMP

dependent protein kinase

Chou & Fassman (1974)• Frequency of occurrence of a given a.a. in α,

β, and loops in all protein structures in the database (statistical)

• Nearest neighbors• output: probability for each residue to be in

α, β, or Loop• Artificial intelligence/neural networks

– Train a computer to recognize patterns – the more information and the “more practice” the higher the accuracy (in progress)

Design

• Minibody• Chymohelizyme• Calcium channel

Minibody• Synthetic (61 a.a.)• All β• Template: heavy chain, variable domain of

IgG• Hypervariable loops• Binding site: Histidines in each

hypervariable loop• Protein it folds and in binds metal (Zn+2)

Chymohelizyme (Science 248: 1544, 1990)

• Design (computer based): 4 helices, parallel, amphipathic, serine protease

• Catalytic TRIAD– Ser, His, Asp at the N-end of the bundle in the

same spatial arrangement as chymotrypsin• “oxyanion hole” and substrate binding pocket

for acetyl tyrosine ethyl ester, a classical substrate of CT were included in the design

• Synthetic enzyme is catalytically active and inhibitor-specific

How can proteins fold so fast?

• Proteins fold to the lowest-energy fold in the microsecond to second time scales. How can they find the right fold so fast?

• It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest-energy one is found (Levinthal’s paradox)

• Search for the minimum is not random because the direction toward the native structure is thermodynamically most favorable

H1AH1

Slide 67

H1 insert figure 4-29a and bHeather, 6/28/2012

AH1 4-29 c and d included--crop?Hug, Alyssa-Rae, 10/26/2012

Proteins folding follow a distinct path

Chaperones prevent misfolding 

Chaperonins facilitate folding