Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

• Published in: The FEBS journal Vol. 272; no. 15; pp. 3960 - 3966
• Main Authors: Su, Zhengding, Wu, Jiun‐Ming, Fang, Huey‐Jen, Tsong, Tian‐Yow, Chen, Hueih‐Min
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.08.2005; Blackwell Publishing Ltd
• Subjects: Amino acids; aromatic amino acid; Bacteria; Biochemistry; Circular Dichroism; Enzyme Stability - physiology; Lysine - chemistry; Lysine - genetics; Lysine - metabolism; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - genetics; Micrococcal Nuclease - metabolism; Mutagenesis, Site-Directed; Protein Folding; Protein Renaturation; Protein Structure, Tertiary; refolding; Spectrometry, Fluorescence; stability; staphylococcal nuclease; Temperature; Tryptophan - chemistry; Tryptophan - genetics; Tryptophan - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2005.04814.x

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 ΔHcal relative to the wild‐type, whereas W140A and W140O display zero enthalpy change (ΔHcal≈ 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.