tailieunhanh - Báo cáo khoa học: Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B

A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent-exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in D 5 -3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side-chains of Leu125 and Val127. | ềFEBS Journal Small exterior hydrophobic cluster contributes to conformational stability and steroid binding in ketosteroid isomerase from Pseudomonas putida biotype B Young S. Yun1 2 Gyu H. Nam1 2 Yeon-Gil Kim1 3 Byung-Ha Oh1 3 and Kwan Y. Choi1 2 1 Division of Molecular and Life Sciences Pohang University of Science and Technology South Korea 2 NationalResearch Laboratory of Protein Folding and Engineering Pohang University of Science and Technology South Korea 3 NationalCRI Center for Biomolecular Recognition Pohang University of Science and Technology South Korea Keywords conformationalstability ketosteroid isomerase small exterior hydrophobic cluster steroid binding surface hydrophobic residue Correspondence K. Y. Choi Division of Molecular and Life Sciences Pohang University of Science and Technology Pohang 790-784 South Korea Fax 82 54 279 2199 Tel 82 54 279 2295 E-mail kchoi@ Database The atomic coordinate and structural factor of W92A have been deposited in the Protein Data Bank under the access code 1W6Y. Received 5 November 2004 revised 26 January 2005 accepted 24 February 2005 doi A structural motif called the small exterior hydrophobic cluster SEHC has been proposed to explain the stabilizing effect mediated by solvent-exposed hydrophobic residues however little is known about its biological roles. Unusually in A5-3-ketosteroid isomerase from Pseudomonas putida biotype B KSI-PI Trp92 is exposed to solvent on the protein surface forming a SEHC with the side-chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI-PI mutants of those amino acids associated with the SEHC were prepared. The W92A L125A V127A and W92A mutations largely decreased the conformational stability while the L125F V127F mutation slightly increased the stability indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased