lectures 6 & 7 -protein foldingclasses.biology.ucsd.edu/bibc100.fa16/documents...protein...
TRANSCRIPT
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