Folding

Databases
Evolution
Classification
Structure viasualization
Sequence alignments
Profiles
Homology modeling
Fold recognition
Protein design
 Suggested books (protein science)

Charles R. Cantor & Paul R. Schimmel

„Biophysical chemistry„ (vol. I, II, III)
W. H. Freeman and Company,
San Francisco, CA

David Eisenberg
"Physical Chemistry"
The Benjamin/Cummings
publishing company, Inc.
Menlo Park, CA

Carl Branden and John Tooze

„Introduction to protein structure„
Garland Publishing, Inc. New York, NY

Thomas E. Creighton
„Proteins„
W. H. Freeman and Company, New York, NY
 Suggested books (bioinformatics)

Durbin et al.
„Biological sequence analysis“
Cambridge University Press

Orengo et al.
„Bioinformatics“
Bios Scientific Publishers Limited

Tramontano
“Bioinformatica”
Zanichelli
 Christian B.
Anfinsen

urea dialysis
native denatured renatured
RNase A βSH RNase A RNase A

specific activity

The thermodynamic hypothesis about protein folding

Anfinsen 1973 Science 181, 223-230
 The Levinthal´s paradox
Protein of 101 amino acids.
3100 = 5 x 1047 configurations.
Sampling 1013 configurations per second (3 x 1020 per year),
It would take 1027 years to try them all.
Proteins fold on a time scale of seconds or less.

=> Hypothesis of obligatory pathways due to kinetic barriers

Is this really a paradox?

(Levinthal 1968 J. Chim. Phys. 65, 44-45)
From obligatory pathways to funnels

(Wolynes et al. 1995 Science 267, 1619-1620)

(Dill & Chan 1997 Nat. Struct. Biol. 4, 10-19)
 Forces

Hydrogen bonds
Van der Waals interactions
Hydrophobic interactions
Electrostatic interactions
Equations

ΔG = ΔG° + RT ln Q
Q = Π i [ Pi ] / Π i [ Ri ]
ΔG° = − RT ln K eq
ΔG = ΔH − TΔS
ΔC p = ∂ T ΔH = T∂ T ΔS
∂ T ln K eq = ΔH ° / RT 2 (van' t Hoff)
T2
ΔH = ∫ C p dT (calorimetry)
T1
Derive as an excercise the
van’t Hoff’s equation...
 U = Q – W (first law of thermodynamics)
Direction of dU = dQ – PdV (constant P, T)
Note: PdV = work
spontaneous H = U + PV
reactions dH = dU + PdV (constant P, T)
dH = dQ

G = H – TS
dG = dH – TdS (constant P, T)

W P surrounding

dSsurrounding = dQsurrounding/T (second law of

system Q
thermodynamics)
dSsurrounding = – dQsystem/T = – dHsystem/T
Universe
dSuniverse = dSsurrounding + dSsystem
dSuniverse = – dHsystem/T + dSsystem
dSuniverse = – dGsystem/T

>0 <0
 Marginal conformational
stability of proteins

ΔG° = ΔH ° − TΔS °

ΔG° = 0.9
ΔH ° = 57
ΔS ° = 0.185
TΔS ° = 56.1

Values (kcal/mol) for RNase A unfolding, pH 2.5, 30 °C

Differential Scanning
Calorimetry
Fatto fino a qui
 Gibbs-Helmholtz equation
ΔC p = ∂ T ΔH ΔC p = T∂ T ΔS
ΔH = TΔC p + c ΔS = ΔC p ln T + c
ΔH m = Tm ΔC p + c ΔS m = ΔC p ln Tm + c
c = ΔH m − Tm ΔC p c = ΔS m − ΔC p ln Tm
ΔH = TΔC p + ΔH m − Tm ΔC p = ΔS = ΔC p ln T + ΔS m − ΔC p ln Tm =
= ΔC p (T − Tm ) + ΔH m = ΔC p ln T / Tm + ΔS m

ΔG = ΔC p (T − Tm ) + ΔH m − TΔC p ln T / Tm − TΔS m =
= ΔH m − TΔS m + ΔC p [(T − Tm ) − T ln T / Tm ]
ΔGm = ΔH m − Tm ΔS m = 0 ⇒ ΔS m = ΔH m / Tm

ΔG = ΔH m (1 − T / Tm ) + ΔC p [(T − Tm ) − T ln T / Tm ]
PGK RNaseA

UV
UV (rev)
Viscosity
ORD

DNaseA

CD
Viscosity
 ΔG N→U from urea denaturation
y
-8 27 istr
8 0 9 9 8 em

06 ,
( 1 ioch

8
63 )
B

Close system (p, T)

ΔG = ΔH – TΔS KN U
→ = [U]/[N] = (εN – ε)/(ε – εU)
ΔG° = – RT ln Keq
εN = ε*N + mN [D]
ε = εN [N]/[T] + εU [U]/[T] εU = ε*U + mU [D]
[T] = [N] + [U] ΔG° = ΔG°* + mG [D]

ε = [(ε*N + mN [D]) + (ε*U + mU [D]) e – (ΔG°*/RT + m G

[D]/RT)]/

/[1 + e – (ΔG°*/RT + m G
[D]/RT)]
Evidence for intermediates

Discrepancy ΔHcal and ΔHvH

Discrepancy different biophysical methods
Two-phase transition
Direct detection by HX or MS