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Thermodynamics of the complex protein unfolding reaction of barstar

Title data

Agashe, Vishwas R. ; Schmid, Franz X. ; Udgaonkar, Jayant B.:
Thermodynamics of the complex protein unfolding reaction of barstar.
In: Biochemistry. Vol. 36 (7 October 1997) Issue 40 . - pp. 12288-12295.
ISSN 0006-2960
DOI: https://doi.org/10.1021/bi971062d

Abstract in another language

The complex unfolding reaction of barstar has been characterized by studying the apparent rate of unfolding, monitored by intrinsic Trp fluorescence, as a function of temperature and guanidine hydrochloride (GdnHCl) concentration. The kinetics of unfolding and folding of wild-type (wt) barstar at 5 degrees C were first studied in detail. It is shown that when unfolding is carried out using concentrations of GdnHCl in the posttransition zone of unfolding, the change in fluorescence that accompanies unfolding occurs in two phases: 30% of the change occurs in a burst phase that is complete within 4 ms, and 70% of the change occurs in a fast phase that is complete within 2 s. In contrast, when the protein is unfolded at 25 degrees C, no burst-phase change in fluorescence is observed. To confirm that a burst-phase change in fluorescence indeed accompanies unfolding at low temperature, unfolding studies were also carried out on a marginally destabilized mutant form of barstar for which the burst-phase change in fluorescence is shown to be as high as 70%. These results confirm a previous report [Nath et al., (1996), Nat. Struct. Biol. 3, 920-923], in which the detection of a burst-phase change in circular dichroism at 222 nm during unfolding at 25 degrees C led to the inclusion of a rapidly formed kinetic intermediate, IU, on the unfolding pathway. To characterize thermodynamically the unfolding pathway, apparent unfolding rates were then measured at six different concentrations of GdnHCl in the range 2.6 to 5.0 M, at five different temperatures from 5 to 46 degrees C. The subsequent analysis was done on the basis of the observation that a preequilibrium between the fully folded state (F) and IU gets established rapidly before further unfolding to the completely unfolded state (U). The results indicate that IU has a specific heat capacity similar to that of F and therefore suggest that IU is as compact as F, with practically no exposure of the hydrophobic core. On the other hand, the transition state of unfolding has a 45% greater heat capacity than F, indicating that significant hydration of the hydrophobic core occurs only after the rate-limiting step of unfolding.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 9315868
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Professorship Biochemistry - Univ.-Prof. Dr. Franz Xaver Schmid
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Professorship Biochemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 18 May 2015 10:06
Last Modified: 18 May 2015 10:06
URI: https://eref.uni-bayreuth.de/id/eprint/13534