Four-State Folding of a SH3 Domain: Salt-Induced Modulation of the Stabilities of the Intermediates and Native State

Dasgupta, Amrita; Udgaonkar, Jayant B.

doi:10.1021/bi300223b

Cited by 17 publications

(43 citation statements)

References 104 publications

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“…The mechanism by which a protein achieves its native structure is much more sensitive to environmental tuning than the final native fold itself. Changes in solvent conditions as well as mutations may cause the folding mechanism to switch between a two‐state and a three‐state transition, via destabilization or stabilization of an intermediate. Alternatively, subtle changes in folding conditions may cause a switch from a pathway on which only the N and U states are populated, to a pathway on which folding occurs via the accumulation of intermediates .…”

Section: Tuning the Cooperativity Of Protein Folding And Unfolding Trmentioning

confidence: 99%

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How cooperative are protein folding and unfolding transitions?

2016

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A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.

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Section: Tuning the Cooperativity Of Protein Folding And Unfolding Trmentioning

confidence: 99%

“…Multi‐step folding is undeniable for many proteins because their folding intermediates are stable enough to accumulate and be easily detectable . In such cases, structural characterization of intermediates provides a wealth of information on folding pathways, and allows different folding pathways to be distinguished from each other .…”

Section: Introductionmentioning

confidence: 99%

How cooperative are protein folding and unfolding transitions?

2016

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“…The DMG is particularly hard to identify in studies of protein folding because it appears to form after the rate-limiting step of folding (8,16). Intermediates that form after the ratelimiting step of folding, however, can be identified from studies of unfolding at high denaturant concentration, and for several proteins (24)(25)(26)(27)(28). In particular, it has been shown for barstar that the same mechanism for unfolding and folding in urea is applicable over the urea concentration range 0 to 7 M (29).…”

Section: Introductionmentioning

confidence: 99%

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Sarkar

Udgaonkar

Krishnamoorthy

2013

Biophysical Journal

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Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N(∗). Fluorescence resonance energy transfer measurements show that N(∗) then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N(∗) and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.

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“…SH3 domains are small globular proteins and are considered to be archetypal of two-state folding proteins . They play important roles in signal transduction, where they provide the interaction surface for multimeric protein complexes. , The SH3 domain of the PI3 kinase was initially characterized as a cooperatively folding protein (two-state folder), , but subsequent equilibrium and kinetic folding studies showed that it (un)folds via multiple intermediate states. − Previous unfolding studies using HX-MS as a probe have shown that the unfolding of the PI3K SH3 domain is a highly heterogeneous process. , The initial structure opening events occur through a continuum of intermediate states. Only at a later stage does the remaining structure unfold in a cooperative manner.…”

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The Osmolyte TMAO Modulates Protein Folding Cooperativity by Altering Global Protein Stability

Jethva

Udgaonkar

2018

Biochemistry

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The folding of many globular proteins from the unfolded (U) to the native (N) state appears to be describable by a two-state N ↔ U model, which has led to the general belief that protein folding occurs in a highly cooperative manner. One reason for the widespread belief in "two-state folding" is that protein folding reactions are invariably studied by ensemble averaging probes and not by probes that can distinguish as well as quantify the multiple conformations that may be present. Consequently, how cooperativity is affected by protein stability, protein sequence, and solvent conditions is poorly understood. In this study, hydrogen exchange coupled to mass spectrometry (HX-MS) of the PI3K SH3 domain was carried out in the presence of a stabilizing osmolyte, trimethylamine N-oxide (TMAO). By showing that HX occurs under the EX1 regime even in the presence of 2 M TMAO, we were able to examine the temporal evolution of the populations of the different conformations present together. A strong link between protein folding cooperativity and protein stability is revealed. Increasing stability is accompanied by an increase in the ruggedness of the free energy landscape as well as diminished cooperativity; the number of amide sites simultaneously opening up their structure decreases with an increase in TMAO concentration. A comparison of the effect of TMAO to that of urea on the intrinsic dynamics of the PI3K SH3 domain indicates that TMAO counteracts the effect of urea not only on protein stability but also on protein folding cooperativity.

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Four-State Folding of a SH3 Domain: Salt-Induced Modulation of the Stabilities of the Intermediates and Native State

Cited by 17 publications

References 104 publications

How cooperative are protein folding and unfolding transitions?

How cooperative are protein folding and unfolding transitions?

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

The Osmolyte TMAO Modulates Protein Folding Cooperativity by Altering Global Protein Stability

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