Effect of a specific hydrogen bond (N138ND 2–Q106O) on conformational integrity, stability, and activity of staphylococcal nuclease

• Published in: Archives of biochemistry and biophysics 2003-12, Vol.420 (1), p.87-94
• Main Authors: Huang, Sun, Yin, Jinhu, Feng, Yanming, Jing, Guozhong
• Format: Article
• Language: English
• Subjects: Conformational integrity; Enzyme Activation; Enzyme Stability; Hydrogen Bonding; Micrococcal Nuclease - chemistry; Micrococcal Nuclease - metabolism; Mutation; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Specific hydrogen bond; Stability; Staphylococcal nuclease; Staphylococcus - chemistry; Staphylococcus - enzymology; Structure-Activity Relationship; Substitution mutation
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2003.09.030

There are two hydrogen bonding interactions (N138ND2–Q106O and Y54OH–S141OG) between the C-terminal region and the main body of staphylococcal nuclease (SNase). To examine the role of these hydrogen bonds, SNase 141 and its three mutants, SNase 141N138D, SNase 141S141A, and SNase 141N138D/S141A, were created. The N138D mutation has the N138ND2–Q106O interaction deleted and the S141A mutation has the Y54OH–S141OG and S141OG–N138O interactions deleted. The conformational features, stability, and activity of the proteins have been compared by using circular dichroism, intrinsic and ANS-binding fluorescence, GdnHCl-induced denaturation, and activity assay. The results clearly show that the N138D mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; in contrast, the S141A mutation has no such effect on structure. These results strongly suggest that the specific hydrogen bond, N138ND2–Q106O, plays an important role in maintaining the conformational integrity and stability of the nuclease.