Differential Salt-induced Stabilization of Structure in the Initial Folding Intermediate Ensemble of Barstar

Pradeep, Lovy; Udgaonkar, Jayant B.

doi:10.1016/s0022-2836(02)01068-9

Cited by 43 publications

(88 citation statements)

References 69 publications

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“…For CDmonitored folding in the absence of osmolyte (Fig. 4d), the values obtained for ⌬G UI and m UI agree with previously determined values (20). For folding data obtained in the presence of either 1 M TMAO (Fig.…”

Section: Equilibrium Urea-induced Unfolding Transitions In the Absupporting

confidence: 90%

“…(20,32). Similar results were obtained when unfolding in TMAO and sarcosine was monitored using far-UV CD at 222 nm ( Fig.…”

Section: Discussionsupporting

confidence: 79%

“…The collapse reaction was, however, observed not to be accompanied by any change in fluorescence (20,24,31), as also seen here in both the absence (Fig. 4a) and the presence of osmolytes (Fig.…”

Section: Osmolytes Do Not Alter the Basic Folding Mechanism-asupporting

confidence: 82%

“…The folding mechanism of barstar has been characterized in detail (14,20,(31)(32)(33). Under strongly stabilizing conditions, the folding of barstar can be represented as follows:…”

mentioning

confidence: 99%

“…In the first of these studies, which was the first demonstration of structural heterogeneity in the folding intermediate of any protein, I E was shown to comprise of at least two structural components that form on competing folding pathways from the same population of U molecules (14). More recent studies indicate that I E is an ensemble of at least three different structural forms, each of which is stabilized differentially, and hence populated differentially, in the absence and presence of salts (20). I L also shows structural heterogeneity (34).…”

mentioning

confidence: 99%

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Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

Pradeep

Udgaonkar

2004

Journal of Biological Chemistry

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Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, I E , on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of I E but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of I E but do so differentially; the fluorescence and circular dichroism properties of I E differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of I E is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the I E ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available.

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Section: Equilibrium Urea-induced Unfolding Transitions In the Absupporting

confidence: 90%

“…(20,32). Similar results were obtained when unfolding in TMAO and sarcosine was monitored using far-UV CD at 222 nm ( Fig.…”

Section: Discussionsupporting

confidence: 79%

Section: Osmolytes Do Not Alter the Basic Folding Mechanism-asupporting

confidence: 82%

“…The folding mechanism of barstar has been characterized in detail (14,20,(31)(32)(33). Under strongly stabilizing conditions, the folding of barstar can be represented as follows:…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

Pradeep

Udgaonkar

2004

Journal of Biological Chemistry

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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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Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein

Bhattacharjee

Udgaonkar

2022

Protein Science

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Distinguishing between competing pathways of folding of a protein, on the basis of how they differ in their progress of structure acquisition, remains an important challenge in protein folding studies. A previous study had shown that the heterodimeric protein, double chain monellin (dcMN) switches between alternative folding pathways upon a change in guanidine hydrochloride (GdnHCl) concentration. In the current study, the folding of dcMN has been characterized by the pulsed hydrogen exchange (HX) labeling methodology used in conjunction with mass spectrometry. Quantification of the extent to which folding intermediates accumulate and then disappear with time of folding at both low and high GdnHCl concentrations, where the folding pathways are known to be different, shows that the folding mechanism is describable by a triangular three-state mechanism. Structural characterization of the productive folding intermediates populated on the alternative pathways has enabled the pathways to be differentiated on the basis of the progress of structure acquisition that occurs on them. The intermediates on the two pathways differ in the extent to which the α-helix and the rest of the β-sheet have acquired structure that is protective against HX. The major difference is, however, that β2 has not acquired any protective structure in the intermediate formed on one pathway, but it has acquired significant protective structure in the intermediate formed on the alternative pathway. Hence, the sequence of structural events is different on the two alternative pathways.alternative pathways, hydrogen exchange, intermediate, kinetics, monellin | INTRODUCTIONA major challenge in the field of protein folding is to detect and characterize structurally the intermediates that populate during folding. The nature of the intermediates and the role they play during folding are not yet fully understood. Several small proteins appear to fold by a two-state mechanism, without populating any intermediates to detectable extents, calling into question the significance of intermediates during folding. [1][2][3][4][5][6][7][8][9] Very Abbreviations: D, deuterium; DcMN, double-chain monellin; ETD, electron transfer dissociation; GdnDCl, guanidine deuterochloride; GdnHCl, guanidine hydrochloride; HX-MS, hydrogen exchange coupled to mass spectrometry; MNEI, single-chain monellin.

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Differential Salt-induced Stabilization of Structure in the Initial Folding Intermediate Ensemble of Barstar

Cited by 43 publications

References 69 publications

Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar

How cooperative are protein folding and unfolding transitions?

Differentiating between the sequence of structural events on alternative pathways of folding of a heterodimeric protein

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