title: Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC.
abstract: The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.
authors.affiliation: Institute of Microbiology, University of Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany.
authors.forename: Nora
authors.initials: N
authors.lastname: Imhof
authors.forename: Andreas
authors.initials: A
authors.lastname: Kuhn
authors.forename: Uwe
authors.initials: U
authors.lastname: Gerken
mesh_terms: D036022:Capsid Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D036022:Capsid Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; C412443:YIDC protein, E coli; D014364:Tryptophan