In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-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 Glu(75) and Lys(9), Tyr(93), and His(121). 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. Study holds ProTherm entries: 12690, 12691, 12692, 12693, 12694, 12695, 12696 Extra Details: beta-barrel; hydrogen bonding; thermal stability; electrostatic interactions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||25|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|