Lecture 7 Protein Folding

James Chou

BCMP201 Spring 2008

Lecture Outline

The problem of protein folding

How proteins fold?

How to predict protein folds?

An observation by Anfinsen and colleagues

Control experiment

Bovine pancreatic RNase

Urea Urea denatures protein mainly by making the
polar and non-polar residues have similar
solubility.

Refolding experiment

Haber and Anfinsen. JBC 1961; 236(2):422-4

. Anfinsen’s Dogma Anfinsen et al. PNAS 1961. 47(9):1309 .

The problem of protein folding ? Amino acid Tertiary sequence structure .

If the protein can sample a new conformation at a rate of 1013 s-1. 65:44 Zwanzig et al. it will take 1027 years to try them all. there are 3100 = 5 x 1047 conformations. Longer than the age of the universe! Therefore. 89:20-22 . Levinthal C. Levinthal paradox Assume each amino acid backbone can be in 3 conformational states. proteins must fold in “pre-arranged pathways” and in a cooperative manner. for 101 residues. PNAS 1992. Extrait du Journal de Chimie Physique 1968..

.The probability of forming contact C2 is much higher if C1 is formed than in the absence of C1. 90:1942-6 . Dill et al.Cooperativity in protein folding : How a globally optimal state can be found without a global search? Origin of cooperativity -. PNAS 1993.

Coil-Helix transition -.the paradigm for cooperativity in biopolymers Nucleation of alpha helix The first H-bond .

# 60.Initiation of a helical turn is much harder than appending another residue to a helical segment. due to higher entropic penalty ! " #60 ! " #40 ! ! " 180. + 60 favored rotamer .

. kb kf equilibrium const for each reaction s = kb !!! A# k f " dA = !k f [ A ] + kb [ B ] = 0 K eq = [ B] k f = !! !B kb dt [ A ] kb .....ccchcccc.#!! f " ! .cccccccc... !k !!! 1st helical turn ..#!! f " ! . kb k !!! 3rd helical turn .........ccchcccc....ccchhhcc.#!! f " ! .ccchhccc.. ! << 1 kb k !!! 2nd helical turn .....ccchhccc.

The Zimm-Bragg-Lifson-Roig theory of coil-helix transition !k k k k k !!! C#!! f " ! H 1 ! #!!f !! " ! H 2 ! #!!f !! " ! $ ! #!!f !! " ! H n "1 ! #!!f !! " ! Hn kb kb kb kb kb !! C#!" ! Hn Kn = [ H ] n = !s n [C] N"3 ! # ( k + 3) ( N " k " 2 ) s k Fraction of residues in helix = k =1 $ N "3 k' . . N &1 + ! # ( N " k " 2 ) s ) % k =1 ( where N is the number of residues in the polypeptide chain.

The temperature dependence of s is given by ln s ∝ −ΔG o k BT . Cooperative transition has a sigmoidal profile N "3 Melting curve of a coiled-coil ! # ( k + 3) ( N " k " 2 ) s k protein in water f = k =1 $ N "3 ' N &1 + ! # ( N " k " 2 ) s k ) % k =1 ( The sharpness of the transition depends both on and N. . being sharper at smaller values of and at larger N.

The process of unfolding shows a bimodal distribution.Cooperativity results roughly in a two-state system Luminescence decay kinetics of Cyt c measured at various times after initial denaturation. . one for largely unfolded state and other includes nearly folded states.

“Structure and Mechanism in Protein Science” . The free energy of protein folding ~50 kJ/mol for 10 kDa protein From Alan Fersht.

. cannot point inside toward the bonds are in the true tetrahedral hydrophobic sphere arrangement. Hydrophobic interaction (entropic) Pure H2O H2O around a hydrophobic molecule Water molecules have less degrees of freedom in the clathrate cage arrangements Water molecules have more because some H-bonds degrees of freedom as the H.

Interfacial Area. More Water Ordered Less Water Ordered . Formation of protein hydrophobic core in water ΔS > 0 Unfolded Folded More Hydrocarbon-Water Less Hydrocarbon-Water Interfacial Area.

Hydrogen bonds (enthalpic) Hydrogen bonds within protein Hydrogen bonds with H2O (hydration) !H of H-bond = " ( 4 to 20 ) kJ mol"1 .

Other Enthalpic Interactions Electrostatic or Coulomb interactions between ions van der Waals interactions Metal coordination Disulfide bonds .

Dissecting the free energy of protein folding !G Unfolded #!!! !! " ! Folded !G = !H " T !S < 0. !G =~ "50 kJ/mol Hydrophobic effect ! T "S ! 0 !G ~ "50 kJ/mol ~ !200 kJ/mol H-bonds !H ! 0 chain conformational " "500 kJ/mol entropy ! T "S ! 0 ~ 750 kJ/mol VDW !H ~ " 50 kJ/mol Electrostatic !H ! "50 kJ/mol .

break H-bonds and other enthalpically favorable interactions !G = !H " T !S < 0 Hydrophobic effect ! T "S ! 0 !G ~ "50 kJ/mol ~ !200 kJ/mol H-bonds chain conformational Heating makes ΔH entropy ! T "S ! 0 less negative ~ 750 kJ/mol VDW Electrostatic . Denaturation by Heat -.

entropy of H2O around non-polar residues is less different H-bonds chain conformational from those around polar entropy ! T "S ! 0 residues ~ 750 kJ/mol VDW Electrostatic .reduce the contribution from hydrophobic effect !G = !H " T !S < 0 !G ~ "50 kJ/mol Hydrophobic effect Near freezing T. Denaturation by Cold -.

“Structure and Mechanism in Protein Science” . Pathways of protein folding From Alan Fersht.

Folding pathway of Barnase Barnase is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. 94:13409-13 . Bond et al.. PNAS 1997.

. JMB 1995.NMR studies of denatured Barnase reveal residual 2nd structures 1H (ppm) Arcus et al. 254(2):305-21 .

Hsp60 and Hsp70 Hsp60 . provide a folding chamber Hsp70 . mainly to block aggregation . Chaperone assisted protein folding Two most important types of chaperones.In bacteria.In all living organisms.

GroEL-GroES chaperone .Hsp60 .

36(2):229-56 . Quarterly Rev of Biophysics 2003.GroEL-GroES chaperone Fenton and Horwich. Hsp60 .

DnaK binds unfolded proteins by recognizing an extended region of the polypeptide chain that is rich in hydrophobic residues substrate .

92:351-66 . free unfolded protein E J J Bukau and Horwich. Cell 1998.

Computational Protein Folding Amino acid Tertiary sequence structure .

4:10-19 . Nature Struct Biol 1997. The free energy landscape of protein folding Ideal funnel Reality funnel Dill and Chan.

dt ri – position of the ith atom mi – mass of the ith atom V – total potential energy of the system The total potential energy is a function of the atomic positions (3N) of all the atoms in the system. A brief note on molecular dynamics simulation The basic idea is to solve Newton’s equation of motion for every atom in the system d 2 ri !" qi V = mi 2 . they must be solved numerically. Due to the complicated nature of this function. there is no analytical solution to the equations of motion above. .

velocities and accelerations can be approximated by a Taylor series expansion: 1 r( t + !t ) = r( t ) + v ( t )!t + a( t )!t 2 2 1 r( t " !t ) = r( t ) " v ( t )!t + a( t )!t 2 2 d 2 ri !"qi V = mi 2 dt Summing the above two equations. . we get r( t + !t ) = 2r( t ) " r( t " !t ) + a( t )!t 2 The Verlet algorithm uses positions and accelerations at time t and the positions from time t-dt to calculate new positions at time t+dt.Example of computer simulation using the Verlet algorithm All the integration algorithms assume the positions.

water and water channel .MD simulation of membrane.

stanford.http://www.Folding@home by the Pande group at Stanford .edu/group/pandegroup/folding/villin/ Game .

Perform MD simulation at each T step.) 2. x. between 0 and 1 # !"G & accept the move if x < exp % $ kBT (' and reject otherwise.Jumping out of the false minima Temperature annealing Melt the system at 2000 C and slowly cool down to 20 C. Metropolis Monte Carlo 1. Make a move (alter the conf. . Calculate ΔG for the move 3. Generate a random number.

. Pure statistical or machine-learning methods are becoming more and more powerful due to the rapidly expanding data base. 2008.Statistical methods for predicting protein structures On Feb 12. there are 48891 Structures in PDB.

… etc. e. pi = % residues of AA type i Generate similar vectors for known classes of folds. from the PDB. coil V β α/β α Chou PY.Amino Acid Composition. .….g. α/β. Prediction of Protein Cellular Attributes Using Pseudo.Early predictions of protein structural properties use amino acid composition The Chou-Fasman Method v = [ p1 . 13(2):222-45. β.. p2 . Fasman GD. α. p20 ]. 43:246-55. (1974). Proteins 2001. Biochemistry. Prediction of protein conformation.

Evidence theory.Knowledge-based methods for predicting protein structures Homology Modeling . Support Vector Machine Combining All You’ve Got .Neural network Pattern Recognition .Rosetta from the Baker Lab .Multiple AA sequence alignment PSI-BLAST (take into account sequence evolution information) Artificial Intelligence .