putting everything together: contributions to protein...

Putting everything together:

Contributions to protein stability (cont’d from lecture 2)

•  How well do these principles account for observed experimental data on protein stability and binding? Unfortunately, because the free energy of protein folding is the difference between two very large contributions: the large chain entropy loss upon folding and the large gain in hydrophobic interactions, it is not currently possible to predict overall protein stability even from high resolution crystal structures.

•  Much more amenable to analysis are the changes in protein stability brought about by single amino acid changes. Studies of the effects of a large number of such sequence changes have led to the general conclusions listed on the next slide. It should be kept in mind, however, that while something may be inferred about effects of the mutations in the native state if a high resolution structure is available, there is considerable uncertainty about the effects of mutations on the denatured state.

ΔG = Gnative - Gdenatured

Experimental Data

(1) Sequence changes at buried sites almost always have much larger effects on stability than sequence changes at exposed sites. The small change at exposed sites is not surprising given that these residues are likely to have similar environments (ie, largely solvated) in both the denatured and native states. (See figure on λ repressor on next slide)

Conclusions From Studies of Protein Stability

(2) Charged residues and to a lesser extent polar residues are disfavored at buried sites. This is expected given the large energetic cost of burying a charge.

(3)  Sequence changes which reduce the amount of hydrophobic burial are destabilizing. (see table on next slide)

Conclusions From Studies of Protein Stability (ctd.)


Substitution ∆ ∆ G (kcal/mol) average

Ile -> Val 1.3+/- 0.4

Ile -> Ala 3.8 +/- 0.7

Leu -> Ala 3.5 +/- 1.1

Val -> Ala 2.5 +/- 0.9

-CH2- 1.2 +/- 0.9

Met -> Ala 3.0 +/- 0.9

Phe -> Ala 3.8 +/- 0.3

(4) Sequence changes which disrupt side chain packing in the interior or leave large cavities are unfavorable.

(5) Salt bridges between oppositely charged residues on the protein surface contribute relatively little to stability, probably because the more favorable electrostatic interactions are offset by the entropic cost of ordering the sidechains. Repulsive interactions between same charged residues on the surface can be quite destabilizing.


Surface area of the cavity

(6) Interactions between negatively charged residues at the N termini of alpha helices with the helix dipole formed by the lining up of all of the

dipoles in the individual peptide bonds are stabilizing (this was first noted by one of the more senior lecturers in this course!)

A new method for predicting the effect of mutations on stability of proteins/protein interfaces is now available on the web: http://robetta.bakerlab.org/ (Interface Alanine Scanning)


References

Most of what has been covered in the lecture on Protein Stability can be found mainly in Creighton, ch4 especially sections 4.1, 4.3 and 4.4. (available on HSB and BIOCHEM libraries)

Protein Folding I

David Baker, October 20th, 2010

Folding I A. Simple example of folding: Lattice model

B. Folding of larger proteins frequently occurs through populated intermediates

Case study: RNaseH

Methods:

Circular Dichroism

Stopped Flow mixing

Hydrogen Deuterium exchange/NMR

Similar intermediates observed in kinetic and equilibrium experiments

C. Folding of small proteins can occur without detectable intermediates.

Case study: Protein L

Methods:

Fluorescence

Hydrogen Deuterium exchange/ MS

Fitting of thermodynamic and kinetic data to 2-state model

X ray solution scattering

D. Single molecule measurements of protein folding and estimation of absolute height of free energy barrier

E. Folding of a de novo designed protein

Simple example of folding: Lattice model

•  1)

useful pedagogically (very concrete)

•  2)

highlight important issues

•  3)

may have some bearing on reality

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•  Model

–  two types of residues:

O X

–  residues restricted to 2-D square lattice:

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–  contact energy ε between adjacent X residues (ε < 0)

P(N) = exp ( -ε/kT ) / Z Z = Σ exp ( - Ei/kT ) = states = 4 exp ( 0/kT ) + exp ( -ε/kT) = 4 + exp ( -ε/kT ) Hence P(N) = 1/ ( 4 exp ( ε/kT ) + 1) as T -> 0 , P(N) -> 1 as T -> infinity , P(N) -> 1/5 (all five states equally probable)

Folding Kinetics in a Lattice Model First, we need a move set:

Given a move set, we can explore possible folding pathways:

Folding of larger proteins Case Study:Folding of Rnase H

–  I. Circular dichroism

•  Differential absorbance of circularly polarized light

–  CD(L) = εL - εR

•  Periodic secondary structure (alpha helices, beta sheets) differentially

absorb circularly polarized light and thus have strong CD

–  II. Stopped flow mixing

•  Most proteins fold on the ms-second time scale. To initiate folding, rapidly mix denatured protein (in guanidine or urea solution) with buffer lacking denaturant

–  III. Hydrogen Deuterium exchange

•  Solvent exposed amide protons can exchange with deuterons

•  Sites of exchange can be identified by NMR or MS.

Different experimental methods may be used to monitor folding:

Circular Dichroism: Probe of Secondary Structure

CD spectra : peptide models of secondary structure elements

CD spectra for typical proteins

The Amide Exchange Experiment (Roder and Baldwin 1988):

To identify which portion of the molecule is structured in the intermediate, use HD exchange Hydrogen Deuterium exchange

Folding of Rnase H (142 residues) I. At very low pH (< 2), a partially folded "molten globule" like state is populated.

CD analysis of this state shows substantial secondary structure (Biochemistry 35, 11951-11958).

II. At equilibrium at neutral pH, similar partially folded states are populated at very low levels. These states are detectable using native state HD exchange (NSB 3, 782-787)

–  Are these partially formed states populated during refolding?

–  Stopped flow CD shows that an intermediate with substantial secondary structure is

formed rapidly after the initiation of refolding:

•  A striking result is that the same part of the protein is ordered

–  1) in the kinetic folding intermediate

–  2) in a well populated equilibrium partially folded state at very low pH

–  3) in very rare partially folded species in equilibrium with the native

state at neutral pH

•  Conclusion: hierarchical formation of structure; regions that are most stable form first.

The Hierarchical Formation of Structure …

–  I. Fluorescence

•  Light at one wavelength absorbed, and light at a longer wavelength emitted

•  The quantum yield (intensity) and the wavelength of the emitted light are sensitive to the environment.

•  The intrinsic fluorescence of tryptophan is particularly useful

–  II. HD exchange/Mass Spectrometry

•  Partially folded species with a subset of protons protected from

exchange will appear as separate peak in Mass Spectrum

•  Deuterate all amide protons in denatured protein, rapidly remove

denaturant and expose to H20 after time Δt

•  Species present:

–  DDDDDDDDDD

(complete folding within Δt)

–  HHHHHHHHHH

(not folded within Δt)

–  DDHDDHHDHD

(partially folded species formed within Δt)

Folding of small protein Case Study:Folding of Protein L

Methods:

Intrinsic Tryptophan Fluorescence can be used to Monitor Protein Folding (Protein L)

The fluorescence of a tryptophan in protein L is much greater in the folded state (black squares) than in the unfolded state (open squares). Circles are for protein L lacking the tryptophan.

Protein Folding Rates can be Measured by Stopped-Flow Fluorescence and CD

Fluorescence: -refolded conditions (closed symbols) -unfolded conditions (open symbols)

CD: -refolded conditions (closed symbols) -unfolded conditions (open symbols)

Refolding monitored by quenched flow HD exchange coupled to mass spectrometry

HD Exchange Experiment Coupled to Mass-Spec (Protein L)

Unfolded conditions

After complete refolding

•  Only two states are significantly populated during folding (U and N)

•  Changes in temperature or [denaturant] only affect relative abundance of U and N.

[denaturant]

Energy

Populaton

U__

0M

N__

99% N

2M

U__ N__

50% N, 50% U

4M

N__

99%U

U__

•  U is unstable in 0M denaturant because of exposed hydrophobic groups; increasing [denaturant] reduces strength of hydrophobic effect.

Two State Model

•  U ↔ N

ΔGfold

•  1) Denaturation profiles fit two state model:

ΔGfold = ΔG0 + m [denaturant] = kT ln [U]/[F]

•  2) in two state model, should observe the same transition behavior

independent of the probe used to monitor the folding reaction.

•  eg.

is not consistent with 2-

state model because a state

with properties different from

either U or N is populated

between 2 and 3M

denaturant.

•  3) Single exponential folding and unfolding kinetics.

k

U → N ([U] + [N] = T)

d[U]/dt = -k[U]

[N] = T - [U] = T ( 1 - e-kt)

Evidence for a 2-State Transition

Radius of Gyration for Protein L under different denaturing conditions

The Radius of Gyration Can Be Measured Using Solution X-Ray Scattering

The kinetics of chain collapse during folding are the same as the kinetics of the overall folding reaction.

Direct Measurement of Rate of Chain Collapse

Triple Exponential Refolding of Top7

0.08

0.085

0.09

0.095

0.1

0.105

0.11

0.001 0.01 0.1 1 10

time (s)

fl.

While almost all single domain naturally ocurring proteins have simple two-state folding transitions and simple exponential kinetics, this is not the case for the novel designed protein: TOP7

An Exception to the Rule

S2-S4 Fragment (red) CFr (red)

-1400000

-1200000

-1000000

-800000

-600000

-400000

-200000

0

0 1 2 3 4 5 6

[gnd]

Mol

ar E

llipt

icity

-14000

-12000

-10000

-8000

-6000

-4000

-2000

0

0 1 2 3 4 5 6 7 8[gnd]

Mea

n R

esid

ue E

liptic

ity

H1-H2 Fragment (red)

-600000

-500000

-400000

-300000

-200000

-100000

0

100000

0 0.5 1 1.5 2 2.5 3 3.5 4

[gnd]

Mol

ar E

liptic

ity

The complex folding kinetics of TOP7 may be due to kinetic traps on its folding landscape

Studying Protein Folding Using Single Molecule Measurement

•  Lack of broadening of FRET efficiency distributions suggest reconfiguration time fast compared to time of pulse (1ms). Upper bound is 25µs.

•  In simple diffusion picture of folding reaction, folding time =2π reconfiguration time * exp (ΔG/kT)

•  Folding time from stopped flow experiments is 12ms

•  Leads to estimate of free energy barrier height of 4-11 kT

Estimate of the Absolute Height of Free Energy Barrier to Folding

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