title: Structural cooperativity in the SH3 domain studied by site-directed mutagenesis and amide hydrogen exchange.
abstract: We have studied the effects produced by site-directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of alpha-spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline-rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen-deuterium exchange (HX) between the wild-type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domain's core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.
authors.affiliation: Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Casares
authors.forename: M
authors.initials: M
authors.lastname: Sadqi
authors.forename: O
authors.initials: O
authors.lastname: López-Mayorga
authors.forename: J C
authors.initials: JC
authors.lastname: Martínez
authors.forename: F
authors.initials: F
authors.lastname: Conejero-Lara
mesh_terms: D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D018909:src Homology Domains
chemical_list: D013049:Spectrin; D006859:Hydrogen; D003903:Deuterium