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J. Biol. Chem., Vol. 276, Issue 49, 46039-46045, December 7, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/49/46039    most recent M106620200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Leung, K. W. Articles by Chen, H. M. Search for Related Content PubMed PubMed Citation Articles by Leung, K. W. Articles by Chen, H. M. Social Bookmarking                      What's this?

Significance of Local Electrostatic Interactions in Staphylococcal Nuclease Studied by Site-directed Mutagenesis^*

King Wong Leung, Yen-Chywan Liaw^§, Siu Chiu Chan, Hau Yi Lo, Faik N. Musayev^§, Jack Z.-W. Chen, Huey-Jen Fang^¶, and Hueih Min Chen^¶

From the ^¶ Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan 115, ^§ Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, and  Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

In this paper, we show that amino acids Glu⁷³ and Asp⁷⁷ of staphylococcal nuclease cooperate unequally with Glu⁷⁵ to stabilize its structure located between the C-terminal helix and -barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD_222 nm. Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu⁷⁵ and Lys⁹, Tyr⁹³, and His¹²¹. In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD_222 nm close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.

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