van t Hoff Analysis - Reed Collegepeople.reed.edu/~glasfeld/Chem391_2015/readings/overheads/L06OH... · Protein G - B1 domain Value of the Hydrophobic Core in Protein Stability Guessing

van’t Hoff Analysis

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

270 275 280 285 290 295 300 305 310 315 320 325 330

T (K)

Nor

mal

ized

sig

nal

Kunfold = 0.18

Kunfold = 2.3

Kunfold = 5.6

Kunfold = 1.0

Kunfold = 0.43

van’t Hoff Plot

T (K) Kunfold 1/T (K-1) ln(K) 290 0.18 .00345 -1.7 295 0.43 .00339 -.83 300 1 .00333 0 305 2.3 .00328 +.84 310 5.6 .00323 +1.7

ln(K) = -15700(1/T)+ 52

∆H˚ = -slope*R = +31 kcal/mol ∆S˚ = intercept*R = +100 cal/molK

Equilibrium Denaturation

slope = “m” (α non-polar SA)

intercept = ∆Gunfold H2O

∆G(urea) = -0.83[urea] + ∆G(H2O)

-3

-2

-1

0

1

2

3

0 1 2 3 4 5 6

[urea] (M)

∆G

unfo

ldin

g0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6

[urea]

Sig

na

l (n

orm

aliz

ed

)

Measure equilibrium constants at each concentration of denaturant, which are used to get ∆Gunfold at each conc.

∆Gunfold = ∆Gunfold - m[urea] [urea] H2O

Protein G - B1 domain

Value of the Hydrophobic Core in Protein Stability

Guessing at Enthalpy Assume each H-bond in 2˚ Structure is 0.5 kcal/mol more energetic than w/ water. 50 H-bond provides ∆Hunfold = +25 kcal/mol stability Guessing at Entropy Assume gain of 9 conformations/residue in unfolding: ∆Sunfold = Rln956 res. = 240 cal/mol•K -T∆Sunfold = -72 kcal/mol Actual ∆Hunfold = +64 kcal/mol ∆Sunfold = +148 cal/mol•K (-T∆S = -42 kcal/mol) vdW contribute bulk of enthalpic stabilization H-phobic effect counters unfavorable entropy of folded protein.

Hole in T4 Lysozyme

Leu99 to Ala Mutant Creates 150 Å3 hole

Matthews (1992) Science 255, 178

∆∆G

unf

Environment Plays A Role in Energetics of H-bonding

Nat. Mol. Struct. Biol., 16, 684 (2009)

PIN1

Environment Plays A Role in Energetics of H-bonding

Nat. Mol. Struct. Biol., 16, 684 (2009)

Replacing a Charged Triad

Disulfide in RNase Sa

C

N

∆Gu = 6.1 kcal/mol + disulfide ∆Gu = 0.3 kcal/mol - disulfide

Cys7-Cy96

Gene 32 Protein: A Structural Zn2+

∆Hunfold = + 139 kcal/mol +Zn2+ ∆Hunfold = + 84 kcal/mol -Zn2+

Biochemistry. 1988 Jul 12;27(14):5240-5.

Distribution of 4˚ Structures

α4 Tetramers

C4 Tetramer D2 Tetramer

α6 Hexamers

C6 Hexamer D3 Hexamer

Hemoglobin - α2β2 Tetramer

Heterotrimeric G Protein

Functionally Distinct Domains

Mammalian Fatty Acid Synthase

Domain Motion - MntR Manganese Transport Regulator

Thanks Wendy Breyer, Kayce Spear

Domain Motion - MntR Manganese Transport Regulator

Thanks Wendy Breyer, Kayce Spear

Tobacco Mosaic Virus