Journal of Molecular Biology
Nucleation and the Transition State of the SH3 Domain
Introduction
Current understanding of the protein folding process is largely based on the theory of nucleation1, 2 and energy landscape theory.3 For the majority of small proteins, which serve as model systems for studying protein folding, the process occurs via a two-state mechanism.4 In analogy to a first-order phase transition, the folded and unfolded states may be thought of as different phases (i.e. liquid and gas) and the transition state (TS) as the “critical nucleus” that seeds the transition5. Since the kinetics of the transformation between the thermodynamically stable folded and unfolded states is determined by the TS, understanding this state allows for a deeper understanding of the rate and mechanism of folding. The folding process may also be thought of statistically with the formation of a transition state ensemble (TSE) as the rate-limiting process in folding.5 Experiments, mainly based on the protein engineering method of “ϕ-value analysis”,6 have made significant progress in testing theories of folding. Concurrently, theory and simulation have continued to improve our understanding of the nature of nucleation in the TSE.7
A prototypical example of the interplay between theory and experiment is the structural interpretation of experimental ϕ-values through simulation.8, 9 This method for reconstructing a protein's TSE from experimental constraints has proven highly useful in understanding folding, especially as it was refined to test the assumption that a set of structures meeting experimental constraints is, in fact, a TSE.10, 11 A TS is characterized by a transmission coefficient of 1/2. In proteins, this corresponds to a probability of folding, or “pfold”5 equal to one half. Verifying this condition prior to making statements regarding computational models of the TSE is absolutely essential as it has been shown that an ensemble of structures meeting a set of ϕ-value restraints is not composed entirely of TS conformations.11, 12 Once a model of the TSE is obtained, it is possible to conduct analyses that are currently impossible by experiment, such as exploring the role of residues for which mutation does not cause sufficient destabilization and building an atom-level structural model of the TSE.
SH3 is a widely studied protein that exhibits two-state folding, and is composed of two orthogonally packed three-stranded β-sheets that form a single hydrophobic core.13 SH3 domains have served as the experimental13, 14 and computational15, 16, 17, 18 model for numerous recent studies of nucleation in protein folding. Although there have been simulations that attempted to reconstruct the TSE using ϕ-values,17, 18 the essential step of testing the assumption that the resulting structures are transition states was not necessarily taken.18 As it has been shown that not all conformations consistent with experimental ϕ-values are members of the TSE, it is difficult to accurately interpret these conclusions regarding nucleation and the TSE. In the following study we use the src, fyn, and spectrin SH3 domain and reported experimental ϕ-values19 as model systems to study the nature of nucleation in the protein folding transition state. Importantly, pfold analysis is used to quantify the position of each structure along the folding pathway. We also examine the role and formation of the hydrophobic core and explore the order of folding events in this classic nucleation-condensation mechanism. In SH3, nucleation is early and separate from hydrophobic core formation. The early, polarized TS that we observe is largely solvent-exposed and is formed by a minimal number of contacts.
Section snippets
A model of the TSE
A side-by-side comparison of the native and transition states is presented in Figure 1. The general features of all observed SH3 TSEs include denaturation of the N and C termini, turns and loops, and a small amount of secondary structures located in the central β strands. The TSE for the three different SH3 domains exhibit average Cα root mean square deviation (RMSD) ranging from 7.1 to 11.4 Å and radii of gyration (Rg) of 12.5 to 14.53 Å, which is largely expanded compared to the ∼10 Å Rg of the
Discussion
We have presented a pfold verified, all-atom model of the TSE in three different SH3 domains. This allows for rigorous conclusions regarding the nature and mechanism of nucleation. In constructing the TSE, residues were restrained, but the way in which the minimal set of restraints was met (contacts between residues) was not predetermined. Importantly, the true TSE was identified through (unconstrained) pfold analysis. It is noteworthy that the set of available ϕ-values for SH3, and other
Constructing and verifying the TSE
Simulations were initiated using coordinates from the crystal structures of the src-SH3 of tyrosine kinase37 (1FMK), fynSH322, 23 (1FYN), and α-spectrin SH324 domains (1BK2). The simulation was propagated by Monte Carlo dynamics using a move set that included rotations of backbone (ϕ-ψ) angles and side-chain (χ) torsion angles, while maintaining planar peptide bonds. All non-hydrogen atoms are represented as impenetrable hard spheres38, 39 and excluded volume is continually enforced. Energy was
Acknowledgements
We thank Dr Brian N. Dominy for assistance with CHARMM, and Eric J. Deeds for his comments on the manuscript. This work was supported by an HHMI pre-doctoral fellowship (I.A.H.).
References (52)
- et al.
Folding nuclei in proteins
FEBS Letters
(2001) - et al.
Theory of protein folding
Curr. Opin. Struct. Biol.
(2004) How do small single-domain proteins fold?
Fold Des.
(1998)- et al.
Pathways for protein folding: is a new view needed?
Curr. Opin. Struct. Biol.
(1998) - et al.
Protein folding funnels: the nature of the transition state ensemble
Fold Des.
(1996) - et al.
Commitment and nucleation in the protein G transition state
J. Mol. Biol.
(2004) - et al.
Stiffness of the distal loop restricts the structural heterogeneity of the transition state ensemble in SH3 domains
J. Mol. Biol.
(2002) - et al.
Thermodynamics and folding kinetics analysis of the SH3 domain form discrete molecular dynamics
J. Mol. Biol.
(2002) - et al.
Formation of the folding nucleus of an SH3 domain investigated by loosely coupled molecular dynamics simulations
Biophys. J.
(2004) - et al.
Similarities between the spectrin SH3 domain denatured state and its folding transition state
J. Mol. Biol.
(2000)
Posttransition state desolvation of the hydrophobic core of the src-SH3 protein domain
Biophys. J.
Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state
J. Mol. Biol.
Direct molecular dynamics observation of protein folding transition state ensemble
Biophys. J.
The folding thermodynamics and kinetics of crambin using an all-atom Monte Carlo simulation
J. Mol. Biol.
Validity of Go models: comparison with a solvent-shielded empirical energy decomposition
Biophys. J.
Does native state topology determine the RNA folding mechanism?
J. Mol. Biol.
How to derive a protein folding potential? A new approach to an old problem
J. Mol. Biol.
Protein folding theory: from lattice to all-atom models
Annu. Rev. Biophys. Biomol. Struct.
Mapping the transition state and pathway of protein folding by protein engineering
Nature
Meeting halfway on the bridge between protein folding theory and experiment
Proc. Natl Acad. Sci. USA
Three key residues form a critical contact network in a protein folding transition state
Nature
Constructing, verifying, and dissecting the folding transition state of chymotrypsin inhibitor 2 with all-atom simulations
Proc. Natl Acad. Sci. USA
Simulation, experiment, and evolution: understanding nucleation in protein S6 folding
Proc. Natl Acad. Sci. USA
Folding dynamics of the src SH3 domain
Biochemistry
Long-range order in the src SH3 folding transition state
Proc. Natl Acad. Sci. USA
Transition states for protein folding have native topologies despite high structural variability
Nature Struct. Mol. Biol.
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Identification of a conserved aggregation-prone intermediate state in the folding pathways of Spc-SH3 amyloidogenic variants
2012, Journal of Molecular BiologyCitation Excerpt :Over the years, these “guinea pigs” of protein folding research have been providing invaluable insight about the principles that govern this remarkable biological process. Indeed, early experimental studies on SH3 domains together with recent computer simulations have been decisive in elucidating the nature of the nucleation–condensation mechanism and to learn about the transition state of protein folding.1–10 More recently, researchers were able to discover the existence of “hidden” intermediates in the folding pathways11 and to understand how nonnative interactions can assist the folding process12–14 by studying mutant variants of Fyn-SH3.
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Parallel Folding Pathways in the SH3 Domain Protein
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2009, Journal of Physics Condensed MatterThe effect of surface tethering on the folding of the src-SH3 protein domain
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