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A kinetic approach to determining the conformational stability of a protein that dimerizes after folding

Title data

Hoffmann-Thoms, Stephanie ; Schmid, Franz X.:
A kinetic approach to determining the conformational stability of a protein that dimerizes after folding.
In: Biochemistry. Vol. 51 (8 May 2012) Issue 18 . - pp. 3948-3956.
ISSN 1520-4995
DOI: https://doi.org/10.1021/bi300154z

Official URL: Volltext

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Abstract in another language

A strongly stabilized form of the β1 domain of the streptococcal protein G, termed Gβ1-M2, was previously obtained by an in vitro selection method for stabilized protein variants. It contains four substitutions, but how they contribute to the Gibbs free energy of denaturation (ΔG(D)) could not be determined, because, unlike the wild-type protein, Gβ1-M2 dimerizes in a spectroscopically silent reaction. Here we determined the ΔG(D) of the folded Gβ1-M2 monomer by using a kinetic approach that uncouples the folding of the monomer from dimerization. The conformational equilibration of the monomer is faster than dimer formation, and therefore, its stability constant could be determined from the ratio of the rate constants for monomer unfolding and refolding. In this approach, double-mixing experiments were essential for uncovering the unfolding kinetics of the transient Gβ1-M2 monomer and the association of the monomers after their folding. The analysis revealed that the selected substitutions stabilize the Gβ1-M2 monomer by 15 kJ mol(-1) in an additive fashion. The combination of single- and double-mixing kinetic experiments thus allowed us to determine the thermodynamic stability of a transient species that is inaccessible in equilibrium experiments. It can be applied for proteins in which monomer folding and oligomerization are kinetically uncoupled.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 22509974
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: 28 Apr 2015 11:07
Last Modified: 28 Apr 2015 11:07
URI: https://eref.uni-bayreuth.de/id/eprint/11165