Intact Disulfide Bonds Decelerate the Folding of Ribonuclease T1

Mücke, Matthias; Schmid, Franz X.

doi:10.1006/jmbi.1994.1408

Cited by 58 publications

(33 citation statements)

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“…All fragments containing domain 1 showed k cat /K m values ranging from 1.25 to 1.6 ϫ 10 6 M Ϫ1 s Ϫ1 for the tetrapeptide succinyl-Ala-Leu-ProPhe-paranitroanilide in both the protease-coupled and the pro- In a second approach, we tested the PPIase activity of FKBP52 fragments in the refolding of a protein. The substrate we used for these measurements was RCM-T1 (37,43,44). This substrate is unfolded in 100 mM Tris, pH 8.0, and refolds spontaneously upon dilution into the same buffer containing 2 M NaCl.…”

Section: Resultsmentioning

confidence: 99%

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Localization of the Chaperone Domain of FKBP52

Pirkl¹,

Fischer²,

Modrow³

et al. 2001

Journal of Biological Chemistry

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FKBP52, a multidomain peptidyl prolyl cis/transisomerase (PPIase), is found in complex with the chaperone Hsp90 and the co-chaperone p23. It displays both PPIase and chaperone activity in vitro. To localize these two activities to specific regions of the protein, we created and analyzed a set of fragments of FKBP52. The PPIase activity toward both peptides and proteins is confined entirely to domain 1 (amino acids 1-148). The chaperone activity, however, resides in the C-terminal part of FKBP52, mainly in the region between amino acids 264 and 400 (domain 3). Interestingly, this domain also contains the tetratricopeptide repeats, which are responsible for the binding to C-terminal amino acids of Hsp90. Competition assays with a C-terminal Hsp90 peptide suggest that the non-native protein and Hsp90 are bound by different regions within this domain.

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Section: Resultsmentioning

confidence: 99%

“…The plasmid carrying the yS54G/ P55N variant of RNase T 1 (RCM-T1) was a kind gift of F. X. Schmid (University of Bayreuth, Bayreuth, Germany). RNase-T 1 was purified and modified as described previously (37). hFKBP12 was a kind gift of Gunther Fischer (Max Planck Institute, Halle, Germany).…”

Section: Methodsmentioning

confidence: 99%

Localization of the Chaperone Domain of FKBP52

Pirkl¹,

Fischer²,

Modrow³

et al. 2001

Journal of Biological Chemistry

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“…Larger proteins with a high hydrophobicity cannot escape from a rapid and possibly nonproductive collapse (54,55). These proteins fold more slowly.…”

Section: Folding Of Cspb Depends Strongly On Solvent Viscositymentioning

confidence: 99%

Diffusion control in an elementary protein folding reaction

Jacob

Schindler

Balbach

et al. 1997

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

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137

163

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The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N ª U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.Most protein chains reach their native conformations during folding rapidly and with high precision, even though the native state is only marginally stable and even though an unfolded protein can adopt very many conformations. Often it is assumed that the folding process is so efficient, because it occurs in two distinct stages (1-4). In the first stage the extended protein chain collapses rapidly into a compact form (often called a molten globule), which is already native-like, but still loosely packed (4, 5). In the second, slow stage the protein chain rearranges to the native state, possibly by a restricted search through the compact conformations. This stage shows a high energy barrier and determines the overall rate of folding. Until recently it was assumed that the rapid formation of compact intermediates is a prerequisite for fast and efficient folding (2,(4)(5)(6)(7)(8).Several small proteins fold extremely fast within a millisecond or even less, but no partially structured intermediates could be detected in these folding reactions (9-17). This seems puzzling. Either these proteins do not follow the two-stage model in their folding, or the initial collapse is so specific that the second stage becomes extremely fast. Thus, the compact intermediate would not accumulate, and the diffusive collapse would become rate-limiting for the entire folding reaction. In this case folding should not follow a monoexponential time course. As a third possibility, chain compaction and crossing of the energy barrier could be tightly coupled in folding. Kramers (18) developed a kinetic model for reactions in which the reactants diffuse together in the rate-limiting step, and he found that the time constants of such processes should depend linearly on the viscosity of the medium. Kramers' theory was used by Karplus and Weaver (19,20) when they formulated the diffusion-collision model for protein folding. Folding reactions that are limited in rate by...

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“…RNase Tl is unfolded even at room temperature when the disulfide bonds are broken, but refolding to a native-like conformation can be induced by adding NaCl [34,35]. Measurements of the dimensions will be performed in future provided that the absence of protein aggregation is also maintained under these conditions.…”

Section: Resultsmentioning

confidence: 99%