Protein Folding BIBC 100

Transcription

1 Protein Folding BIBC 100

2 The Folding Problem How Proteins Fold? Consider a protein with 100 a.a 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 years or seconds Cosmic Term: longer than the life of earth/universe

3 Levinthal s Paradox However, in vivo, proteins fold in 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.

4 Second Genetic Code: Sequence Computer Algorithm Folding Structure Design (reverse folding) Input Output Folding Sequence Structure Design Structure Sequence

5 Importance? 1. Too many sequences and still few structures ( , genome) (~10 5 ) Understanding seq.-struct. relation requires solving: Too many structures or The Folding Problem 2. Biotechnology unleashed power Design: drugs, hormones, sensors, processes (photosynthesis), imagine

6 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

7 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

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10 Ribonuclease Refolding Experiment 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

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16 Physics of Folding Entropy drives towards this HB exposed Disorder in a system 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

17 Steps of Folding < ms Up to 1s Unfolded bury core 2 o Molten globule 3 o 4 o protein HB aa (loose 3 o ) (breathing)

18 Energy Funnel for Folding Multiple folding pathways can occur Model this with energy funnel

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20 H2 AH2 N denotes the native fold and is the lowest free-energy state

21 Slide 20 H2 AH2 insert figure 4-29a and b Heather, 6/28/ c and d included--crop? Hug, Alyssa-Rae, 10/26/2012

22 Physical Forces H bond (local, near neighbors) Hydrophobic (compactness/molton globule, distant neighbor) Folding Pathways Funnels Explore the energy landscape or conformational space (degrees of freedom) Proc. Natl. Acad. Sci. USA 89: (1992)

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26 Protein folding follows a distinct path

27 Proteostasis Maintenance of cellular protein activity is accomplished by the coordination of many different pathways

28 Protein misfolding is the basis of numerous human diseases

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31 Computational Modeling Major area of research Infancy We still cannot accurately fold proteins by computer Needed: 1. Understanding process 2. Defining the minimum 3. Faster computers 4. Models testable by experimentation That s why folding and design are two different formulations of the same problem

32 In Vivo Folding Goal: To prevent aggregation (collapsed intermediates) and alternatively folded states Chaperones bind to incompletely folded polypeptides Prevent aggregation Regulate translocation Foldases catalyze folding Native Intermediate Folding N I U chaperones Rx s: -Disulfide Bonds -x-pro peptide bonds -cis-trans isomers

33 Why won t it fold? Most common obstacles to a native fold: Aggregation Non-native disulfide bridge formation Isomerization of proline

34 Chaperones prevent misfolding

35 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

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37 GroEL 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

38 GroES Cap to the GroEL Each subunit sheet hairpin (roof) Mobile loop (int w/ GroEL) 7 subunits in functional molecule

39 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

40 1. GroEL/ATP complex at side A 2. Bind GroES on this side 7 ATP 7 ADP this side has a wider cavity but closed top other side has smaller cavity and open top 3. Side B ring binds unfolded protein GroES falls off of side A ADP falls off of side A 4. Side B ring binds 7 ATPs 5. GroES binds GroEL/ATP 7 ATP 7 ADP protein folding occurs 6. Side A ring binds 7 ATPs protein folding occurs 7 ATP 7 ADP (side A) 7 ADP & GroES (side B) falls off 7. Side A ring binds next unfolded protein

41 Chaperonins facilitate folding The two chambers alternate in binding and folding of client proteins

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43 Mechanism of Chaperonin Function 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

44 GroEL-GroES trapped encapsulating a folding intermediate Cell 153, , June 6, 2013

45 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

46 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-C a 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

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48 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

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50 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 candidates d. Site-selective mutations e. Protein synthesis f. Assay: example T4 lysozyme (x-ray structure known) Reducing degrees of freedom (entropy) increases protein stability

51 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) -Gly Ala usually increase -Pro Ala usually decrease

52 Protein Stability Cont 3. Dipolar stability Helix: N-end (-a.a.) C-end (+a.a.) increase stability by mutating residues at N-end of helices from polar to negative (e.g. ASN ASP, SER ASP)

53 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 Ile Val or Phe Leu Cavity for a CH2 Stability by 1kcal/mol -More delicate design -Needs structure

54 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

55 A prokaryotic Kv channel with essential sequence conservation of the voltage sensor. The Rockefeller University Press Santos J S et al. J Gen Physiol 2006;128:

56 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

57 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)

58 Design Minibody Chymohelizyme Calcium channel

59 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 )

60 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

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67 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

68 H1 AH1

69 Slide 67 H1 AH1 insert figure 4-29a and b Heather, 6/28/ c and d included--crop? Hug, Alyssa-Rae, 10/26/2012

70 Proteins folding follow a distinct path

71 Chaperones prevent misfolding

72 Chaperonins facilitate folding

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