experimental methods for the study of protein...

Experimental methods for the study of protein folding

October 12, 2018 JBB 2026H: Protein Structure, Folding and Design

Lecture 5 Julie Forman-Kay & Hue Sun Chan

Lecture based partly on the slides prepared by Patrick Farber

Protein Folding: A fundamental biophysical problem

Peptide synthesis unfolded

chaperone

misfolding

Goal: Develop means to characterize the fundamental biophysical process of protein folding

folded

Aggregation/fibrils

Hydrophobic Effect

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

Arg Lys

Asp Glu

Asn Gln

Ser

Gly

His

Thr

Ala

Pro Try

Val

Me

t

Cys

Leu

Ph

e Ile Trp

ΔG

tran

sfer

(oct

ano

l)

The hydrophobic effect is the major driving force for protein folding. It is entropically more favorable for water to hydrogen bond with itself than with non-polar amino acid side chains. The hydrophobic effect can be quantified by measuring the partition coefficients of non-polar molecules between water and non-polar solvents.

Salt Bridges – Stabilizing?

http://en.wikipedia.org/wiki/Salt_bridge_(protein_and_supramolecular)

Large de-solvation energetic penalties for burying polar or charged residues Honig & Hubbell (1984) estimated that the cost of transferring a salt bridge from water to the protein environment is approximately 10-16 kcal/mol!

Estimating the Energy of a Salt Bridge - Double mutant analysis

J. Mol. Recognit. 2004; 17: 1–16

If two charges residues are not interacting with each other, then the change in protein stability resulting from the removal of both charges by mutation will be equal to the sum of the stability changes see with the two single mutations. By contrast if the two charges are interacting with each other, the change in stability of the double mutant will not be the sum of the changes. A double mutant cycle yields the coupling free energy of a salt bridge.


Substitution of a salt bridge network with hydrophobic residues stabilizes the domain without significantly changing the conformation The desolvation penalty due to the burial of polar and charged groups in the protein interior (a low dielectric environment) during protein folding, may not be fully recovered by favorable electrostatic interactions in the folded state Salt bridges may contribute to structural specificity, by remaining buried but not packing optimally

Nature Structural Biology 2, 122 - 128 (1995)

Arc repressor of bacteriophage P22 is a homodimeric DNA binding protein Contains a buried salt bridge network containing – R31 (37% solvent accessible) – D36 (0%), and R40 (27%)


Most salt bridges are stabilizing, just not as stable as hydrophobic interactions!

Kumar and Nussinov J Mol Biol. 1999 Nov 12;293(5):1241-55.

Slow folding: Proline Cis-Trans Isomerization

fast

trans slow

cis

trans

trans/cis isomerization

(un)folding

In 1973, Garel & Baldwin discovered that unfolded ribonuclease (RNase) A consists of a mixture of molecules that differ vastly in their rates of refolding. The respective fast-folding (U F) and slow-folding (Us) species coexist in a slow equilibrium and give rise to parallel fast (in the time range of milliseconds) and slow (in the time range of minutes) refolding reactions. UF and Us species have since been detected for many other proteins

Prolyl Isomerase: Enzymatic Catalysis of Slow Protein-Folding Reactions Annual Review of Biophysics and Biomolecular Structure Vol. 22: 123-143 (1993)

Two-state folding The reversible unfolding of small single-domain proteins is usually a cooperative process, in which only the fully native (N) and fully denatured (D) molecules are populated at equilibrium, which can be described by a two-state model:

Nku

kf

¾ ®¾¬ ¾¾ D

Where ku is the unfolding rate, and kf is the folding rate.

KU

=Déë ùû

Néë ùû

DGF-U

= -RT ln KU( ) = -RT ln

ku

kf

æ

èç

ö

ø÷ = DG

D-¹+ DG

¹-N

Detection Methods in Protein folding studies

Early events in Protein Folding, Chemical Reviews 2006 Vol 106 No 5

Detection Methods- 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

Detection Methods- Intrinsic Fluorescence

Equilibrium Unfolding Measurements

KU

=Déë ùû

Néë ùû

=fD

1- fD

DGF-U

= -RT ln KU( )

m= slope Dependence of ΔG on denaturant concentration

DG = DG H2O( ) -m ureaéë ùû

m-values and solvent accessible surface area (ASA)

m-values quantify the dependence of ΔG on denaturant concentration. The relationship between m-values and ΔASA suggests that there is a size (length) dependence on unfolding

Folding rates agree well with contact order

CO =1

L ×NDSi, j

N

åN is the total number of contacts, dSij is the sequence separation in residues between contacting residues i and j and L is the total number of residues in the protein

Rapid Mixing Techniques for Protein Folding Studies - Turbulent Mixing

Time-resolved data are essential for elucidating the mechanism of protein folding. The effects of temperature and/or denaturant concentration give insight into the activation energies and solvent accessibility of the transition state ensemble. In order to obtain kinetic information, rapid mixing techniques are commonly used. These techniques rely on turbulent mixing to achieve complete mixing in a small volume. However, turbulent mixing is slower than laminar flow

If a molecule has a diffusion constant of 1x10-9 m2/s, it will take about 1ms to diffuse over a distance of 1 um (t = r2/D). This means the mechanical mixing must occur in a space of less than 1 micron to achieve sub-millisecond mixing times. This is a large contribution to the dead time of a measurement.

Stopped-flow kinetics

http://www.hi-techsci.com/techniques/stoppedflow/

In a typical stopped flow experiment, a few hundred microliters of solution are delivered to the mixer via two syringes driven by a pneumatic actuator or stepper motors. Flow rates are in the range of 5-15 mL/s with channel diameters of the order of 1mm to ensure turbulent flow conditions. After delivering a volume sufficient to purge and fill the observation cell with freshly mixed solution, the flow is stopped abruptly when a third syringe hits a stopping block or a valve is closed.

Sosnick et al. Proteins (1996) vol. 24 (4) pp. 427-432

Kinetics can be measured by many different spectroscopic techniques, as listed earlier After mixing, structural changes are monitored, and can be observed on the millisecond timescale Curves are fit to an exponential function, depending on the mechanism, and rates are extracted

Continuous Flow techniques

In a continuous flow experiment, the reaction is again triggered by turbulent mixing. The progress of the reaction is sampled under steady state flow conditions as a function of the distance downstream from the mixer.

Adapted from slide prepared by Alan Davidson

(treatment of experimental baselines)

[cf. Slide #13]

slide prepared by Alan Davidson

Chevron Plot

ln kobs( ) = ln k fH2 0( ) - m f denaturant[ ]( )

+ ln kuH2 0( ) + mu denaturant[ ]( )

Zarrine-Afsar and Davidson. The analysis of protein folding kinetic data produced in protein engineering experiments. Methods (2004) vol. 34 (1) pp. 41-50

• The kinetic data from stopped flow or continuous flow experiments are plotted in a chevron curve

• The mf and mu values determined from fitting chevron plots express the linear dependence of ln(kf) and ln(ku) on the concentration of denaturant.

• These parameters can be related to the solvent accessibility of the folding transition state.

Folding Intermediates – Non-two state behavior

Nkni

kin

¾ ®¾¬ ¾¾ Ikid

kdi

¾ ®¾¬ ¾¾ D

pN =kDIkNI

kDIkIN + kIDkIN + kDIkNIpI =

kIDkIN

kDIkIN + kIDkIN + kDIkNIpD =

kDIkNI

kDIkIN + kIDkIN + kDIkNI

Many proteins fold via an intermediate(s). Folding intermediates are typically lowly populated states.

Curved Chevron Plots

Brockwell and Radford. Intermediates: ubiquitous species on folding energy landscapes?. Current Opinion in Structural Biology (2007) vol. 17 (1) pp. 30-7

Mutational Analysis – Specific Amino Acid Dependence on Folding?

• Mutations change the folding rate k and stability

GN–D

• Most proteins are very tolerant to conservative mutations (G

or A), and it does not affect the folded structure.

• Mutations enable residue-specific probing of interactions

important in the transition state of protein folding

• Using kinetic unfolding studies, we can get kf, ku, for each

mutant, and compare the thermodynamic and kinetic

parameters to that of the wild type.

DDG f®u = -RT lnk fmut

k fWT

æ

èçö

ø÷- RT ln

kUWT

kumut

æ

èçö

ø÷

Kinetic m values m values calculated from the kinetic data can be compared with the m values calculated from equilibrium experiments. Significant alterations in the transition state structure caused by any of the mutants examined can result in changes in the mku or mkf values, which are influenced by the total solvent accessibility of the transition state. However, most of the mutants display mku or mkf values that are close to wild-type. alpha values can be calculated from mku and mkf, which reflect the total surface burial of the transition state as a proportion of the total buried in the native state

The Brønsted equations are: Where ko

f and kou are the rate constants for folding and unfolding of the wild-type

protein and βf is a constant related to the degree of structure formation in the transition state. Brønsted plots can used to compare the kinetic behaviors of single amino acid substitutions at different positions in a protein OR multiple mutations at the same position to determine if mutations possess the same degree of partial structure formation in the transition state.

Brønsted Plots – Rate-equilibrium Free Energy Plots

Phi Value Analysis

Φ= 0 Φ= 1

F =DDG

¹-D

DDGN®D

=DG

¹®D

WT - DG¹®D

nut

DGN®D

WT - DGN®D

mut

A Φ‐value close to 1 indicates that the free‐energy change

introduced by the mutation is almost identical for the

transition state and the native state.

A Φ‐value close to 0 indicates that the height of the Gibbs free‐energy

barrier of the transition state was not altered by the mutation. In this

case, the mutated residue does not form native contacts in the transition

state

DDG¹-D = DG¹®D

mut - DG¹®D

wt = -RT lnk fmut

k fwt

æ

èçö

ø÷

DDGN®D = -RT lnkNmut

kNWT

æ

èçö

ø÷- RT ln

kDWT

kDmut

æ

èçö

ø÷

Φ-Value Analysis

For many proteins, the transition state appears to be an expanded form of the native state. - Intermediate Φ-values are interpreted to mean that native

interactions are partially formed in the transition state - m values indicate that transition state structure is quite compact

(70% as compact as native for SH3 domain, 90% as compact as native for cold shock protein)

Some transition state structures are highly polarized, meaning one part of the protein may form earlier in the folding reaction than another Transition state structures are compact but very unstable.

Φ-Value Analysis- CI2

Chymotrypsin Inhibitor 2 (CI2) was one of the first proteins extensively studied by Φ- value analysis. Extensive mutational analysis allows for insight into the transition state structure and mechanism of folding

Classical mechanisms for 2-state folding

Nucleation Condensation mechanism- CI2

• The nucleation condensation mechanism unites features of both the hydrophobic collapse and heirarchical mechanisms.

• Nucleation-condensation invokes the formation of long range and other native hydrophobic interactions in the transition state to stabilize the otherwise weak secondary structure.

• The transition state forms stable folds owing to a combination of long-range tertiary interactions and secondary structure.

• Isolated elements of repeating secondary structure, such as α-helices or β-hairpins, tend to have weak conformational preferences in the absence of the rest of the protein, and are stabilized by tertiary interactions that are made with the rest of the protein.

• The transition state resembles a distorted form of the native structure, with the least distorted part being loosely defined as the nucleus and the distortion tending to increase with increasing distance from the nucleus

Folding Pathway of CI2 An MD unfolding simulations from the native state N to the denatured state(s) D at

225°C shown in reverse. The structures are colored from red at the N terminus to blue at the C terminus.

Fersht, Daggett, Cell, Volume 108, Issue 4, 2002, 573 - 582

Transition state structure from Simulation and Phi-Value Analysis


a, The secondary structure elements are: beta1 (residues 7–13), alpha1 (residues 22–33), beta2 (residues 36–42), beta3 (residues 46–53), alpha2 (residues 55–56), beta4 (residues 77–85), betaT (residues 93–97); residues between these regions are parts of loops. b, the key residues 11, 54 and 94 found from the transition-state analysis are shown as gold spheres on the native structure. They are connected by gold-coloured bonds to the residues (white and black spheres) forming a native contact with them (Y11 has long-range contacts with 47–52 and 78–81, P54 with 5–7 and 34–35, and F94 with 26–31, 36–39 and 50–52). Residues forming the transition-state core as black spheres. They fall into two groups in spatial proximity; there is a larger group (residues 28, 35–39, 51–54 and 90–95) comprising parts of alpha1, beta2, alpha2 and betaT, and a smaller group (residues 11–13, 47 and 78, 79) comprising parts of the beta1, beta3 and beta4 strands in the native structure.

acylphosphatase (AcP)

Vendruscolo et al Nature 409, 641-645


a, The six most representative conformations of the TSE generated from the structure calculations. The three key residues forming the structural core are shown as gold spheres and secondary structural elements are shown as ribbons with arrows indicating the directionality of the beta-strands. Preliminary results for molecular dynamics structures obtained with an all-atom potential and the same phi-value restraints indicate that somewhat more secondary structure, particularly for the helices, is present in the TSE). b, The average structure of the transition state (thick lines) derived from the six conformations shown in a (thin lines). The average structure is obtained by superimposing the three key residues (gold spheres) for all the structures in the TSE. c, Average r.m.s. deviation from the native state as a function of the residue number; the native secondary structural elements are indicated above the diagram.

Vendruscolo et al Nature 409, 641-645

Mapping Folding upon Binding using Phi-Value analysis


Giri R et al. PNAS 2013;110:14942-14947


Φ-value analysis on the binding between pwtKIX and c-Myb*. Measured Φ-values are mapped on the 3D structure of the complex. Following Fersht and Sato, weak, 0 < Φ < 0.3 are represented in red; medium, 0.3 < Φ < 0.7 are represented in magenta; high, 0.7 < Φ < 1 are represented in blue. Φ-values > 1, reporting misfolding events, are indicated in green. A and C refer to tertiary and secondary Φ-values, respectively (as defined in Table 1), whereas in B the structure of bound c-Myb is highlighted in rainbow coloring.

Giri R et al. PNAS 2013;110:14942-14947


Leffler (Bronsted) plot for the binding between pwtKIX and c-Myb*. Each individual point refers to a site directed mutant, as reported in Table1.

Giri R et al. PNAS 2013;110:14942-14947

Hydrophobic Collapse versus Specific Packing in the Transition State

Hydrophobic Collapse versus Specific Packing in the Transition State

R= 0.89 R= 0.83 R= 0.76 R= 0.58

Two-parameter model

T N N H HG G G

Corresponds to both hydrophobic burial and specific packing interactions that are shared by the native and transition states.

xH > 0, nonspecific hydrophobic interactions play a proportionally larger role in the stability of the transition state than they do in the native state

(χH+ χN) ≈ fractional burial of the side chain in the transition state

Two-Parameter fits of Folding Kinetics

• Folding kinetic data for multiple mutations at individual hydrophobic core positions can be well accounted for by a two parameter model

• Formation of native-state like contacts lag behind hydrophobic burial in the transition state for side chains at many positions

What if mutation changes the energetics of the unfolded state?

What if mutation changes the energetics of the unfolded state?

NTL9 – small two-state folding protein

• Two residues can be linked through either

the native state or the DSE or both.

• ΔG°EAB

, ΔG°EA

, ΔG°EB

, and ΔG°E represent

the unfolding free energies of wild type, a

single mutant at site B, a single mutant at

site A, and the double mutant, respectively.

• A negative coupling free energy represents

either an unfavorable interaction in the

native state or a favorable interaction in the

DSE or a combination of both.

• A negative value will also arise if a

favorable DSE interaction is larger than a

favorable native state interaction and vice versa.

Cho J et al. PNAS 2014;111:12079-12084

K12 is energetically coupled to hydrophobic core residues.

Cho J et al. PNAS 2014;111:12079-12084

Double-mutant cycle analysis reveals significant coupling in the DSE which is modulated by

mutation.

Cho J et al. PNAS 2014;111:12079-12084

Couplings are large in the wild type, but are significantly reduced in mutants which weaken DSE hydrophobic clustering. (A) I4 and K12 measured in wild-type and in the L35A backgrounds, (B) A22 and K12 measured in wild-type and in the L47A backgrounds, and (C) A36 and K12 measured in wild-type and the A42G backgrounds.

Modulation of DSE energetic impacts the K12M Φ-value.

Cho J et al. PNAS 2014;111:12079-12084

• Energetically significant coupled interactions can be present in the DSE of globular proteins and can be altered by mutation

• DSE effects can complicate double-mutant cycle studies, and impact the analysis of native state coupling networks; DSE effects could even lead to misleading conclusions about long-range coupling interactions in the native state.

• For the mutations examined here, a double-mutant cycle analysis that ignores DSE effects would lead to the conclusion that there was significant long-range coupling in the native state between K12 and a large number of hydrophobic residues

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