Spectroscopic Characterization of Staphylococcal Nuclease Mutants with Tryptophan at Internal Sites

Tryptophan (Trp) is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far-and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type (WT). Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.National Key Basic Research Program of China [2013CB921904, 2013CB328701-2013CB328706]; National Natural Science Foundation of China [11074016, 11121091, 61177020, 11134001, 10828407]SCI(E)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)ARTICLEwangsf@pku.edu.cn4048701-1-048701-53