title: Probing the subtle conformational state of N138ND2-Q106O hydrogen bonding deletion mutant (Asn138Asp) of staphylococcal nuclease using time of flight mass spectrometry with limited proteolysis.
abstract: Recent studies indicate that the N138ND2-Q106O hydrogen bonding deletion in staphylococcal nuclease significantly alters the conformational integrity and stability of the nuclease. To find out the structural basis of the changes, mass spectrometry and limited proteolysis methods were combined to probe the subtle conformational changes in the SNaseN138D mutant and SNaseN138D-Ca2+-pdTp complex. The results reveal that the N138ND2-Q106O hydrogen bonding deletion makes the C-terminal part of alpha-helix 1 and alpha-helix 2 in the C-terminal subdomain of SNaseN138D unfold to some extent, but does not have much effect on the N-terminal part of alpha-helix 1, alpha-helix 3, and the N-terminal beta-barrel subdomain of SNaseN138D. Binding of ligands makes the alpha-helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state. This study also demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China.
authors.forename: Sun
authors.initials: S
authors.lastname: Huang
authors.forename: Xiajuan
authors.initials: X
authors.lastname: Zou
authors.forename: Peng
authors.initials: P
authors.lastname: Guo
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhong
authors.forename: Jiarou
authors.initials: J
authors.lastname: Peng
authors.forename: Guozhong
authors.initials: G
authors.lastname: Jing
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012697:Serine Endopeptidases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D008836:Micrococcal Nuclease; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase