tailieunhanh - Báo cáo khoa học: Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding

Staphylococcalnuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of pro-tein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in ther-mal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lowerDHcal values than the wild-type protein | ềFEBS Journal Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding Hueih-Min Chen1 Siu-Chiu Chan1 King-Wong Leung1 Jiun-Ming Wu1 Huey-Jen Fang1 and Tian-Yow Tsong2 3 1 Institute of BioAgriculturalSciences Academia Sinica Taipei Taiwan 2 Institute of Physics Academia Sinica Taipei Taiwan 3 Department of Biochemistry University of Minnesota College of BiologicalSciences St. Paul MN USA Keywords staphylococcal nuclease local stability key acidic amino acid unfolding Correspondence . Chen Institute of BioAgricultural Sciences Academia Sinica Taipei Taiwan Fax 886 2 2788 8401 Tel 886 2 2785 5696 extn 8030 E-mail robell@ Received 3 May 2005 revised 3 June 2005 accepted 13 June 2005 doi Staphylococcal nuclease is a single domain protein with 149 amino acids. It has no disulfide bonds which makes it a simple model for the study of protein folding. In this study 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in thermal denaturation experiments we identified two mutants E75G and E129G having approximately 43 and 44 respectively lower AHcal values than the wild-type protein. Furthermore two mutants E75Q and E129Q were created and the results imply that substitution of the Gly residue has little influence on destabilization of the secondary structure that leads to the large perturbation of the tertiary protein structure stability. Two local stable areas formed by the charge-charge interactions around E75 and E129 with particular positively charged amino acids are thus identified as being significant in maintenance of the three-dimensional structure of the protein. Staphylococcal nuclease SNase is a globular protein that consists of 149 amino acids with a molecular mass of about 16 kDa. This protein lacks disulfide bonds .