The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates

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

doi:10.1006/jmbi.1994.0196

Cited by 105 publications

(122 citation statements)

References 29 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…Initial Collapse of the Polypeptide Chain-Early fluorescence resonance energy transfer as well as 8-anilino-1-naphthalenesulfonic acid binding studies had indicated that the polypeptide chain of barstar collapses to a compact form within the initial few milliseconds of refolding in low as well as in marginally destabilizing concentrations of denaturant (14,33), suggesting that (i) the collapse reaction must have brought together hydrophobic residues to form exposed hydrophobic patches capable of binding 8-anilino-1-naphthalene-sulfonic acid (14,33) and (ii) the collapse must occur not only in strongly stabilizing but also in marginally stabilizing conditions for folding, as well. 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.…”

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

confidence: 99%

“…Earlier studies had indicated that I E is compact and possesses solvent-exposed hydrophobic patches (14). In marginally stabilizing conditions (1-1.2 M guanidine HCl), no specific structure is found to form at a few milliseconds of folding, suggesting either that I E was devoid of specific structure (33) or that I E does not accumulate significantly under these folding conditions.…”

mentioning

confidence: 97%

“…Such effects of osmolytes on protein folding pathways have become important to study, because recent studies of the products of the submillisecond folding reactions of several proteins, including barstar (14), ribonuclease A (15,16), lysozyme (17), cytochrome c (18), and apomyoglobin (19), suggest that these early intermediates are structurally heterogeneous. So far, this heterogeneity has manifested itself in two or three co-existing forms (14 -21).…”

mentioning

confidence: 99%

“…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%

“…There is strong evidence for structural heterogeneity in I E , from several studies. 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).…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Pradeep

Udgaonkar

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

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

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

“…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%

See 3 more Smart Citations

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

Pradeep

Udgaonkar

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange

Bhuyan

Udgaonkar

1998

Proteins

Self Cite

View full text Add to dashboard Cite

Equilibrium amide hydrogen exchange studies of barstar have been carried out at pH 6.7, 32 degrees C using one- and two-dimensional nuclear magnetic resonance. An unusually large fraction of the backbone amide hydrogens of barstar exchange too fast to be measured, and the exchange rates of only fifteen slow-exchanging amide sites including indole amides of two tryptophans could be measured in the presence of 0 to 1.8 M guanidine hydrochloride (GdnHCl). Measurement of exchange occurring in tens of seconds in the unfolding transition region was possible by the use of a fast stopped-flow mixing method. The observed exchange rates have been simulated in the EX2 limit according to a two-process model that incorporates two exchange-competent states: a transiently unfolded state (U*) in which many amide hydrogens are completely accessible to solvent-exchange, and a near-native locally unfolded state (N*), in which only one or a few amide hydrogens are completely accessible to solvent-exchange. The two-process model appears to account for the observed exchange behavior over the entire range of GdnHCl concentrations studied. For several measurable slow-exchanging amide hydrogens, the free energies of production of exchange-competent states from the exchange-incompetent native state are significantly higher than the free-energy of production of the equilibrium unfolded state from the native state, when the latter is determined from circular dichroism- or fluorescence-monitored equilibrium unfolding curves. The result implies that U*, which forms transiently in the strongly native-like conditions used for the hydrogen exchange studies, is higher in energy than the equilibrium-unfolded state. The higher energy of this transiently unfolded exchange-competent state can be attributed to either proline isomerization or to the presence of residual structure. On the basis of the free energies of production of exchange-competent states, the measured amide sites of barstar appear to define two structural subdomains--a three-helix unit and a two-beta-strand unit in the core of the protein.

show abstract

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

et al. 1998

View full text Add to dashboard Cite

It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.

show abstract

The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates

Cited by 105 publications

References 29 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

Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

Contact Info

Product

Resources

About