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

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13 Scopus citations


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
Issue number49
StatePublished - Dec 7 2001


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