Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone

Shastry, M.C.R.; Agashe, Vishwas R.; Udgaonkar, Jayant B.

doi:10.1002/pro.5560030907

Cited by 48 publications

(115 citation statements)

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“…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: 81%

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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-asupporting

confidence: 81%

“…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:…”

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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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“…However, Pro isomerization was suggested to occur with ϭ 212 s in stopped-flow folding experiments on Thermus thermophilus RNase H (42), which is two orders of magnitude slower than the transitions observed here. In other proteins, Pro isomerization also was observed on the hundreds of seconds timescale (43)(44)(45). Proteins under strongly denaturing solvent conditions are frequently taken as the ''random coil'' reference state in folding (41).…”

Section: D [5]mentioning

confidence: 99%

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Kuzmenkina

Heyes

Nienhaus

2005

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

173

172

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Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfoldedstate ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Fö rster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of Ϸ20 s at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of Ϸ2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.fluorescence resonance energy transfer ͉ guanidinium chloride ͉ protein folding ͉ RNase H ͉ single-molecule spectroscopy P roteins are complex systems that can exist in a huge number of different conformations. Even in the properly folded, native state, the polypeptide chain adopts many slightly different conformations that can be depicted as local minima in a rugged energy landscape, and relaxations and fluctuations in the landscape are crucially involved in functional processes (1, 2). Conformational heterogeneity is even more of a concern in studies of protein-folding reactions because of the vast number of possible arrangements of the unfolded polypeptide chain and the many complex pathways leading from the ensemble of unfolded to the native conformations in an overall funnel-shaped energy landscape (3-7).Because of Anfinsen's key observation that the native fold is already encoded in the sequence of amino acids (8), the proteinfolding problem has attracted enormous attention, and the field has progressed in a healthy interplay between theory and experiment. A wide variety of biophysical approaches were developed to follow the dynamics of the polypeptide en route to the native state. Valuable insights have been achieved by timeresolved spectroscopic experiments on bu...

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“…The supposedly third phase of proteins, the molten globules, is populated mainly by secondary structures and remnants of tertiary interactions. Moreover, extremes in conditions such as pH, ionic strength, temperature and denaturants would readily induce the equilibrium molten globule state in a protein [36,37,38,39]. Kinetic intermediates have been identified on the folding pathway of many proteins [40,41,42,43] in native-like conditions [39].…”

Section: Discussionmentioning

confidence: 99%

Kinetic analysis of urea-inactivation of β-galactosidase in the presence of galactose

Chilaka

Nwamba

2008

Journal of Enzyme Inhibition and Medicinal Chemistry

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The effect of galactose on the inactivation of purified b-galactosidase from the black bean, Kestingiella geocarpa, in 5 M urea at 508C and at pH 4.5, was determined.Lineweaver-Burk plots of initial velocity data in the presence and absence of urea and galactose were used to determine the relevant , the microscopic rate constants for the free enzyme and the enzymesubstrate complex, respectively. Plots of k þ0 and k 0 þ0 vs. galactose concentrations showed that galactose protected the free enzyme and not the enzyme-substrate complex against urea inactivation via a noncompetitive mechanism at low galactose concentrations and a competitive pattern of inhibition at high galactose concentrations. The implication of the different modes of inhibition in protecting the free enzyme was discussed.

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Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone

Cited by 48 publications

References 40 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

Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions

Kinetic analysis of urea-inactivation of β-galactosidase in the presence of galactose

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