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A 15 N-labelled peripheral-subunit binding domain (PSBD) of the dihydro-lipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assem-bly of the pyruvate dehydrogenase multienzyme complex ofBacillus stearo-thermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the 15 N and 1 H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int. . | ềFEBS Journal Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus Use of a truncated protein domain in NMR spectroscopy Mark D. Allen R. William Broadhurst Robert G. Solomon and Richard N. Perham Cambridge Centre for Molecular Recognition Department of Biochemistry University of Cambridge UK Keywords pyruvate dehydrogenase protein-protein interaction NMR spectroscopy multienzyme complex protein domains Correspondence R.N. Perham Department of Biochemistry University of Cambridge Sanger Building Old Addenbrooke s Site 80 Tennis Court Road Cambridge CB2 1GA UK Fax 44 1223 338707 Tel 44 1223 338635 E-mail r.n.perham@joh.cam.ac.uk Received 19 July 2004 revised 27 September 2004 accepted 28 September 2004 doi 10.1111 j.1432-1033.2004.04405.x A 15N-labelled peripheral-subunit binding domain PSBD of the dihydro-lipoyl acetyltransferase E2p and the dimer of a solubilized interface domain E3int derived from the dihydrolipoyl dehydrogenase E3 were used to investigate the basis of the interaction of E2p with E3 in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearo-thermophilus. Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the 15N and 1H amide chemical shifts when the PSBD forms a 1 1 complex with E3int. All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III. 15N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168 with limited structure present in residues Asn126 to Arg128. The presence of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD. Comparison of the crystal structure of the PSBD bound to E3 Mande SS Sarfaty S Allen MD Perham RN Hol WGJ 1996 Structure 4 277-286 with the solution structure of uncomplexed PSBD described here indicates that the PSBD .
