Increasing Stability Reduces Conformational Heterogeneity in a Protein Folding Intermediate Ensemble

Sridevi, K.; Lakshmikanth, G. S.; Krishnamoorthy, G.; Udgaonkar, Jayant B.

doi:10.1016/j.jmb.2003.12.083

Cited by 54 publications

(66 citation statements)

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“…In the strongly stabilizing conditions provided by the osmolyte, I E is compact with significant structure and hence appears to be the product of a specific folding reaction. A multi-site, time-resolved fluorescence resonance energy transfer characterization (35) of the slow folding reaction has also indicated that different pathways dominate under different folding conditions, when different components of the late intermediate I L are stabilized under different conditions. Very recently, a comparative study of the folding of barstar in urea and guanidine HCl has suggested that different folding pathways are utilized in the two denaturants (29).…”

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

confidence: 99%

“…I L also shows structural heterogeneity (34). Most recently, the use of a multisite time-resolved fluorescence resonance energy transfer approach has shown that the extent of structural heterogeneity depends on how stable the folding conditions are and that different structural components predominate in I L under different folding conditions (35). Obviously, barstar is a good model system for exploring the effects of osmolytes on the structural heterogeneity that now appears characteristic of protein folding reactions.…”

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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: Osmolytes Do Not Alter the Basic Folding Mechanism-amentioning

confidence: 99%

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confidence: 99%

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

Pradeep

Udgaonkar

2004

Journal of Biological Chemistry

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“…Much is known about the folding mechanism of barstar (24)(25)(26)(27)(28)(29)(30), and earlier studies with millisecond time resolution had indicated, albeit indirectly, that the initial, sub-ms folding reaction of barstar is a gradual process (8,9). A very early collapsed structure-less globule, U C , gets transformed into a structured early intermediate, I E (9,25,26), at a few milliseconds of folding.…”

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Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Sinha

Udgaonkar

2008

Proc. Natl. Acad. Sci. U.S.A.

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To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.noncooperative ͉ submillisecond protein folding

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“…A funnel describes a protein's conformational heterogeneity-Conformational heterogeneity has been found in the few experiments that have been designed to look for it (16,143,157,206,209,244). For example, using time-resolved FRET with four different intramolecular distances, Sridevi et al (206) found in Barstar that (a) that the chain entropy increases as structures become less stable, (b) that there are multiple folding routes, and (c) that different routes dominate under different folding conditions.…”

Section: Proteins Fold On Funnel-shaped Energy Landscapesmentioning

confidence: 99%

“…For example, using time-resolved FRET with four different intramolecular distances, Sridevi et al (206) found in Barstar that (a) that the chain entropy increases as structures become less stable, (b) that there are multiple folding routes, and (c) that different routes dominate under different folding conditions. Moreover, changing the denaturant can change the dominant pathway, implying heterogeneous kinetics (143).…”

Section: Proteins Fold On Funnel-shaped Energy Landscapesmentioning

confidence: 99%

Protein Folding Problem

2008

Encyclopedia of Public Health

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The "protein folding problem" consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first.

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Increasing Stability Reduces Conformational Heterogeneity in a Protein Folding Intermediate Ensemble

Cited by 54 publications

References 54 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

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein

Protein Folding Problem

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