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Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions

Title data

Wunderlich, Michael ; Martin, Andreas ; Schmid, Franz X.:
Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions.
In: Journal of Molecular Biology. Vol. 347 (15 April 2005) Issue 5 . - pp. 1063-1076.
ISSN 0022-2836
DOI: https://doi.org/10.1016/j.jmb.2005.02.014

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The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 15784264
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: 27 Apr 2015 07:59
Last Modified: 10 Jun 2015 14:30
URI: https://eref.uni-bayreuth.de/id/eprint/10827