Effect of Salt on the Urea-Unfolded Form of Barstar Probed by <i>m</i> Value Measurements

Pradeep, Lovy; Udgaonkar, Jayant B.

doi:10.1021/bi049320b

Cited by 23 publications

(23 citation statements)

References 63 publications

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“…46 Moreover, mutational studies by Pradeep and Udgaonkar showed that the replacement of charged residues by neutral amino acid residues changes the energetics of the unfolded state of barstar. 47 Finally, the idea of electrostatic repulsions at high globule densities is supported by our experiments at 1.4 ± 0.2 M urea in the presence of different concentrations of KCl (Fig. 5a-c).…”

Section: Discussionsupporting

confidence: 66%

Coulomb Forces Control the Density of the Collapsed Unfolded State of Barstar

Hofmann¹,

Golbik²,

Ott

et al. 2008

Journal of Molecular Biology

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Section: Discussionsupporting

confidence: 66%

Coulomb Forces Control the Density of the Collapsed Unfolded State of Barstar

Hofmann¹,

Golbik²,

Ott

et al. 2008

Journal of Molecular Biology

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“…Several recent studies have shown that charge‐reversal mutations on the surface of proteins can be used to improve electrostatic interactions and increase protein stability (Grimsley et al 1999; Loladze et al 1999; Spector et al 2000; Pradeep and Udgaonkar 2004). In a previous study (Pace et al 2000), we showed that the E74K RNase Sa variant was 1.1 kcal/mol more stable that WT RNase Sa, but the D17K variant was 1.1 kcal/mol less stable than WT RNase Sa.…”

Section: Discussionmentioning

confidence: 99%

Charge–charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

et al. 2005

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Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.Keywords: protein folding; protein stability; folding kinetics; denatured state; charge-charge interactions Protein stability is an important consideration in protein engineering. There is considerable interest in developing methods to increase the stability of a protein. Two ways this can be achieved are (1) decrease the free energy of the native state, and/or (2) increase the free energy of the denatured state. In an earlier study , a charge-reversal mutation (D17K) was made to improve electrostatic interactions on the surface of native ribonuclease Sa (RNase Sa) to stabilize the protein. However, the mutant was less stable than the wildtype protein by about 1 kcal/mol. We suggested that this resulted because electrostatic interactions were more favorable in the denatured state of the mutant than in wild-type RNase Sa, and that this might influence the folding kinetics. The results in the present work support this idea, and show that charge-charge interactions in the transition and denatured state ensembles of a protein can exert a substantial effect on the kinetics of protein folding. Recent work on another protein from Raleigh's lab has reached a similar conclusion (Cho et al. 2004;Horng et al. 2005), and thus these findings may be fairly general. ResultsTo monitor the folding and unfolding of WT RNase Sa and the charge-reversal variant, D17R, by fluorescence spectroscopy, a Trp residue was added in place of Tyr 81. The crystal structure of Y81W showed that the Trp is 87% buried, and the variant is 0.4 kcal/mol less stable than WT (Alston et al. 2004). For Y81W RNase Sa, there is a 2.5-fold increase in fluorescence intensity at 319 nm when the protein folds (Alston et al. 2004). Throughout this work, the Y81W variant will be referred to as WT* and the charge-reversal variant as WT*(D17R). Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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“…This distance is significantly smaller than the end-to-end distance of a 25-aa residue-long polypeptide chain in a random coil conformation, which is estimated to be Ϸ37 Å (33). Hence, a decrease in the D-A distance from the dry molten globule state to the U state appears possible only if some residual structure forms in the U state (34)(35)(36). Indeed, the observation that the W4-C97TNB distance, which separates 93 residues in the sequence, is only Ϸ 26Å in the U state suggests that the U state is not a random coil but is compact.…”

Section: Expansion and Contraction Of Intramolecular Distance During mentioning

confidence: 99%

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

Jha

Udgaonkar

2009

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

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124

176

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Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin ( Two models are commonly invoked to describe the rate-limiting step during protein unfolding. The commonly accepted model is that the rate-limiting step is controlled by the extensive rearrangement of native structure upon the entry of water into the hydrophobic core (1-5). In the alternative model, based on the dry molten globule hypothesis (6), the rate-limiting step is an initial concerted rupture of the tight side-chain packing in the interior, without any entry of water. The free energy barrier arises because the loss of enthalpy in the dry globular transition state has not yet been compensated for by a gain in conformational entropy (7,8). Experimental evidence in support of this hypothesis is, however, scarce and moreover, indirect (9-11). The dry molten globule model posits that the tight packing interactions are lost cooperatively when thermal fluctuations cause secondary structural elements to move marginally apart from each other (7). But small displacements of individual secondary structural units have not been detected as the initial steps of the unfolding of any protein. In particular, the detection of the rotation or translation of an ␣-helix or the fraying movement of a ␤-strand during the formation of a dry molten globule, which would constitute the most direct evidence in support of the dry molten globule hypothesis, has been difficult to capture in experiments.Multisite FRET measurements allow determination of the displacements of specific segments of a protein structure, during folding or unfolding (12)(13)(14)(15)(16)(17)(18)(19). In this study, 2-site FRET measurements of the unfolding of single-chain monellin (MNEI), in both equilibrium and kinetic unfolding experiments, have been carried out to determine how an intramolecular distance, spanning the Nand C-termini of the sole helix in the protein changes, compared with an intramolecular distance corresponding to the end-to-end distance of the protein. MNEI is an intensely sweet, small plant protein in which the sole ␣-helix is packed against a 5-stranded ␤-sheet in a ␤-grasp fold (Fig. 1A). In earlier studies, the folding and unfolding of MNEI have been characterized in detail (20)(21)(22). The folding and unfolding reactions of MNEI appear to be multistate, with multiple intermediates populating on parallel pathways (21). It is shown here that the unfolding of the protein begins with an initial expansion of the protein into a dry molten globular st...

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Effect of Salt on the Urea-Unfolded Form of Barstar Probed by m Value Measurements

Cited by 23 publications

References 63 publications

Coulomb Forces Control the Density of the Collapsed Unfolded State of Barstar

Coulomb Forces Control the Density of the Collapsed Unfolded State of Barstar

Charge–charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

Direct evidence for a dry molten globule intermediate during the unfolding of a small protein

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