We investigated the role of W140 in the folding of Staphylococcal nuclease. For this purpose, we constructed the 19 possible substitution mutations at residue 140. Only three mutants, W140F, W140H, and W140Y, adopted native-like structures under physiological conditions and showed native-like enzymatic activities. In contrast, the other 16 mutants took on compact unfolded structures under physiological conditions and the enzymatic activities of these mutants were decreased to approximately 70% of wild-type levels. These 16 mutants maintained substrate-induced foldability. These results strongly indicate that the side-chain information encoded by residue 140 is essential to maintain a stable native structure, and that this residue must be an aromatic side chain. The order of thermal stability was wild type > W140H > W140F = W140Y. Therefore, the five-membered nitrogen-containing ring of the indole is thought to bear the essential information. In the crystal structure of staphylococcal nuclease, the five-membered ring is at the local center of the C-terminal cluster through hydrophobic interactions. This cluster plays a key role in the interaction connecting the C-terminal region and the N-terminal beta-core. Mutants other than W140H, W140F, and W140Y lost the ability to form the local core, which caused the loss of the long-range interactions between the C-terminal and N-terminal regions. Inhibitor or substrate binding to these mutants compensates for the lack of long-range interactions generated by W140. Study holds ProTherm entries: 18695, 18696, 18697, 18698 Extra Details: protein folding; substitution mutant; sequence-structure relationship;,structure-function relationship; X-ray solution scattering; circular dichroism; hydrolysis of DNA
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:50 p.m.
|Number of data points||11|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|