tailieunhanh - Báo cáo khoa học: Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coliK1 was cloned, expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coliK1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. | ễFEBS Journal Protein folding intermediates of invasin protein IbeA from Escherichia coli Damodara R. Mendu1 Venkata R. Dasari2 Mian Cai1 and Kwang S. Kim1 1 Department of Pediatrics Division of Infectious Diseases Johns Hopkins University Schoolof Medicine Baltimore MD USA 2 Department of Biomedicaland Therapeutic Sciences College of Medicine University of Illinois Peoria IL USA Keywords acid and Gdm-HCl-induced unfolding Escherichia coli molten globule protein unfolding intermediates of IbeA Correspondence K. S. Kim Division of Pediatric Infectious Diseases Johns Hopkins University School of Medicine 200 North Wolfe Street Room 3157 Baltimore MD 21287 USA Fax 1 410 614 1491 Tel 1 410 614 3917 E-mail kwangkim@ Received 9 October 2007 revised 15 November 2007 accepted 28 November 2007 doi IbeA of Escherichia coli K1 was cloned expressed and purified as a His6-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects along with equilibrium unfolding of IbeA were studied in solution. The far-UV CD spectrum of IbeA at pH has a strong negative peak at 215 nm indicating the existence of p-sheet-like structure. The acidic unfolding curve of IbeA at pH shows the existence of a partially unfolded molecule molten globule-like structure with p-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid ANS binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH IbeA exists in a partially unfolded state with characteristics of a molten globule-like state and the protein is in extended b-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative contrary to the cooperativity seen with the native protein suggesting the presence of two domains possibly in the molecular .