Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Jemth, Per; Gianni, Stefano; Day, Ryan; Li, Bin; Johnson, Christopher M.; Daggett, Valerie; Fersht, Alan R.

doi:10.1073/pnas.0401732101

Cited by 99 publications

(150 citation statements)

References 30 publications

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“…The kinetics of a two step reaction should be fitted to the two roots of a quadratic equation, as previously shown for Im7 [28], TT cyt c 552 [29]and the FF domain [30]. In many cases, however, only one relaxation rate constant can be experimentally observed, which jeopardizes a quantitative curve fitting.…”

Section: Identification and Characterization Of Folding Intermediatesmentioning

confidence: 99%

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Identification and characterization of protein folding intermediates

Gianni

Ivarsson

Jemth

et al. 2007

Biophysical Chemistry

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In order to understand the mechanism by which a polypeptide chain folds into its functionally active native state it is necessary to characterize in detail all the species accumulated along the pathway.The elusive nature of protein folding intermediates poses their identification and characterization as an extremely difficult task in the protein folding field. In the case of small single domain proteins, the direct measurement of the thermodynamics and structural parameters of protein folding intermediates has provided new insights on the nature of the forces involved in the stabilization of nascent protein structures. Here we summarize some of the experimental approaches aimed at the detection and characterization of folding intermediates along with a discussion of some general structural features emerging from these studies.

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Section: Identification and Characterization Of Folding Intermediatesmentioning

confidence: 99%

“…The folding of c 552 from Thermus thermophylus [29] and Hydrogenobacter thermophilus [36], the FF domain [30] and Im7 protein [28] In all cases, global analysis of observed kinetics led to the conclusion that the observed folding intermediate was an on-pathway species on the route between the denatured and native states.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Identification and characterization of protein folding intermediates

Gianni

Ivarsson

Jemth

et al. 2007

Biophysical Chemistry

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“…Although some small proteins are thought to fold by a 2-state mechanism in which the unfolded state transitions in a highly cooperative manner to the folded conformer [20] (Figure 1a), it is becoming increasingly clear that for many proteins, folding involves the formation of one or more transiently formed intermediates [1,[6][7][8][9][10]21] (Figure 1b). Sufficiently stable intermediates can be detected kinetically using stop-flow or continuous-flow techniques and proven to be onpathway by kinetic modeling [8,9,22] be populated to a level that is detectable at equilibrium by thermodynamic experiments, and in amenable cases be proven to be on-pathway by relaxation dispersion NMR, a technique which will be described in detail in later sections.…”

Section: Transiently Formed On-pathway Intermediates In Protein Foldingmentioning

confidence: 99%

“…Sufficiently stable intermediates can be detected kinetically using stop-flow or continuous-flow techniques and proven to be onpathway by kinetic modeling [8,9,22] be populated to a level that is detectable at equilibrium by thermodynamic experiments, and in amenable cases be proven to be on-pathway by relaxation dispersion NMR, a technique which will be described in detail in later sections.…”

Section: Transiently Formed On-pathway Intermediates In Protein Foldingmentioning

confidence: 99%

“…Levinthal"s solution to the paradox: protein folding is not a random process, but instead proceeds by some directed mechanism [5]. Indeed, it appears that many of the proteins whose folding has been studied to date follow directed pathways involving the transient accumulation of specific intermediate states with defined structure [1,[6][7][8][9][10]. In principle, structural studies of folding intermediates would provide a wealth of information about the folding process [1,10] and potentially also shed light as to how proteins misfold [11].…”

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Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

et al. 2011

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We have recently reported the atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 [Korzhnev, D. M.; Religa, T. L.; Banachewicz, W.; Fersht, A. R.; Kay, L.E. Science 2011, 329, 1312–1316]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints of the invisible intermediate state measured using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the database structure determination program, CS-Rosetta. As a cross-validation of the structure so produced, we present here the solution structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by mutagenesis that destabilizes the native state. The relaxation dispersion/CS-Rosetta structures of the intermediate are within 2 Å of those of the mimic, with the nonnative interactions in the intermediate also observed in the mimic. This strongly confirms the structure of the FF domain folding intermediate, in particular, and validates the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

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Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

Morris

Searle

2012

CP Protein Science

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We present an overview of the current experimental and theoretical approaches to studying protein folding mechanisms, set against current models of the folding energy landscape. We describe how stability and folding kinetics can be determined experimentally and how this data can be interpreted in terms of the characteristic features of various models from the simplest two-state pathway to a multi-state mechanism. We summarize the pros and cons of a range of spectroscopic methods for measuring folding rates and present a theoretical framework, coupled with protein engineering approaches, for elucidating folding mechanisms and structural features of folding transition states. A series of case studies are used to show how experimental kinetic data can be interpreted in the context of non-native interactions, populated intermediates, parallel folding pathways, and sequential transition states. We also show how computational methods now allow transient species of high energy, such as folding transition states, to be modeled on the basis of experimental Φ-value analysis derived from the effects of point mutations on folding kinetics.

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Demonstration of a low-energy on-pathway intermediate in a fast-folding protein by kinetics, protein engineering, and simulation

Cited by 99 publications

References 30 publications

Identification and characterization of protein folding intermediates

Identification and characterization of protein folding intermediates

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

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