tailieunhanh - Báo cáo khoa học: Substitution of residues at the double dimer interface affects the stability and oligomerization of goose d-crystallin

d-Crystallin is the major structural protein in avian and reptilian eye lenses, and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study, the roles of the side chains of Glu267, Lys315, and Glu327, which provide hydrogen bonds at the double dimer interface, were investigated. Hydrophobic side chain sub-stitution led to all mutant proteins having an unstable dimer interface. | Substitution of residues at the double dimer interface affects the stability and oligomerization of goose ô-crystallin Chih-Wei Huang1 2 z Chih-Chao Tseng1 z Ya-Huei Chen1 Yu-Hou Chen3 Wei-Yuan Chou1 and Hwei-Jen Lee1 1 Department of Biochemistry NationalDefense MedicalCenter Taipei Taiwan 2 Department of Pharmacy Practice Tri-Service General Hospital Taipei Taiwan 3 Genomics Research Center Academia Sinica Taipei Taiwan Keywords argininosuccinate lyase conformational stability 8-crystallin double dimer subunit interface Correspondence H. J. Lee Department of Biochemistry NationalDefense MedicalCenter No. 161 Sec. 6 MinChuan E. Road Neihu Taipei 114 Taiwan Fax 886 2 87923106 Tel 886 2 87910832 E-mail hjlee@ These authors contributed equally to this work Received 26 May 2009 revised 30 June 2009 accepted 10 July 2009 doi Ỗ-Crystallin is the major structural protein in avian and reptilian eye lenses and confers special refractive properties. The protein is a homotetramer arranged as a dimer of dimers. In the present study the roles of the side chains of Glu267 Lys315 and Glu327 which provide hydrogen bonds at the double dimer interface were investigated. Hydrophobic side chain substitution led to all mutant proteins having an unstable dimer interface. The E267L E327L mutant had the greatest sensitivity to temperature urea and guanidinium hydrochloride denaturation and the most extensive exposure of hydrophobic patches as judged by 1-anilinonaphthalene-8-sulfonic acid fluorescence CD and tryptophan fluorescence. In contrast the E267L K315L E327L mutant showed higher stability than the E267L E327L mutant. Some level of the dissociated dimeric form was observed in the K315L mutant but it was not observed for the K315A and E267L K315L mutants. The E327L mutant was partially in the dissociated dimeric form whereas the E267 E327L mutant was predominantly dissociated into dimers. In contrast the triple mutant of E267L .