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PubMedDate not supplied

[I87E mutation prevents barstar dimerization].

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) x 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 investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

Bacillus↗

PubMedDate not supplied

[Residual dipolar couplings and molecular dynamic calculations as a source for refinement of protein spatial structures].

The precision of techniques and factors affecting the interpretation of residual dipolar couplings (RDCs) in analysis of spatial structures of partially aligned proteins are discussed. Experimental RDC values were obtained for pairs of 1H-15N nuclei of the protein barstar partially aligned in a liquid crystalline matrix of bicelles composed of dimiristoylphosphatidylcholine and dihexanoylphosphatidylcholine. The observed couplings agree well with the spatial structures of barstar determined earlier by X-ray and NMR methods. However, the differences between the experimental and calculated RDCs that were calculated on the basis of the known spatial structures of barstar, exceed the experimental errors three- to fourfold. These discrepancies can be explained by differences in the protein structures in solution and in crystal, a limited precision of the X-ray analysis, and the intramolecular mobility of the protein molecule. A comparison of the results of modeling of the molecular dynamics of barstar in solution, crystal structures, and the experimental RDCs showed that the methods of molecular dynamics provide for a reasonable description of the character and amplitudes of internal motions and they should be considered for the correct determination of protein spatial structures from NMR spectroscopic data.

Amino Acid Substitution↗