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, Hueih Min Chen

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

In this paper, we show that amino acids Glu73 and Asp 77 of staphylococcal nuclease cooperate unequally with Glu 75 to stabilize its structure located between the C-terminal helix and β-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 CD222 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 CD222 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.

Original languageEnglish (US)
Pages (from-to)46039-46045
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number49
DOIs
StatePublished - Dec 7 2001

Fingerprint Dive into the research topics of 'Significance of Local Electrostatic Interactions in Staphylococcal Nuclease Studied by Site-directed Mutagenesis'. Together they form a unique fingerprint.

  • Cite this

    Leung, K. W., Liaw, Y. C., Chan, S. C., Lo, H. Y., Musayev, F. N., Chen, J. Z. W., Fang, H. J., & Chen, H. M. (2001). Significance of Local Electrostatic Interactions in Staphylococcal Nuclease Studied by Site-directed Mutagenesis. Journal of Biological Chemistry, 276(49), 46039-46045. https://doi.org/10.1074/jbc.M106620200