title: Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding.
abstract: Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point-mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins (positions 1-139 [SNase(1-139) or W140O] and positions 1-141 [SNase(1-141) or E142O]) were generated. Differential scanning microcalorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged DeltaH(cal) relative to the wild-type, whereas W140A and W140O display zero enthalpy change (DeltaH(cal) approximately 0). Far-UV CD measurements indicate secondary structure in W140A but not W140O, and near-UV CD measurements indicate no tertiary structure in either W140 mutant. These observations indicate an unusually large contribution of W140 to the stability and structural integrity of SNase.
authors.affiliation: Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC.
authors.forename: Zhengding
authors.initials: Z
authors.lastname: Su
authors.forename: Jiun-Ming
authors.initials: JM
authors.lastname: Wu
authors.forename: Huey-Jen
authors.initials: HJ
authors.lastname: Fang
authors.forename: Tian-Yow
authors.initials: TY
authors.lastname: Tsong
authors.forename: Hueih-Min
authors.initials: HM
authors.lastname: Chen
mesh_terms: D002942:Circular Dichroism; D004795:Enzyme Stability / Q000502:physiology; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014364:Tryptophan; D008836:Micrococcal Nuclease; D008239:Lysine