Authors: D. S. Korchuganov¹; A. A. Schulga¹; Ya. S. Ermolyuk¹; V. A. Mitkevich²; M. Ya. Reibarkh¹; S. B. Nolde¹; A. A. Makarov³; A. S. Arseniev⁴; M. P. Kirpichnikov¹

Source: Russian Journal of Bioorganic Chemistry, Volume 30, Number 6, November 2004 , pp. 577-581(5)

Publisher: MAIK Nauka/Interperiodica

Abstract:

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 ± 1.1) × 10^–11 M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in the investigations where the protein dimerization is undesirable.

Keywords: barnase; barstar; protein dimerization

Document Type: Research article

DOI: http://dx.doi.org/10.1023/B:RUBI.0000049775.11808.a1

Affiliations: 1: Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia 2: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia. The Center for Medical Studies at Moscow, University of Oslo, ul. Vavilova 34/5, Moscow, 119991 Russia 3: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia 4: Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia aars@nmr.ru, Email: aars@nmr.ru

Publication date: 2004-11-01

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