Substrate-induced conformational change of the Escherichia coli membrane insertase YidC.
The membrane insertase YidC from Escherichia coli reversibly binds its substrate Pf3 coat protein. The effect of this initial binding process was examined in vitro by fluorescence quenching of the tryptophan (Trp) residues of YidC which are highly sensitive fluorescent probes for changes of the protein's tertiary structure. Membrane-reconstituted (in DOPC or DOPE/DOPG vesicles) as well as detergent-solubilized (C(12)PC) YidC was titrated with a Trp-free Pf3 coat mutant. Quenching of the intrinsic Trp fluorescence after titration indicates a change in the tertiary structure of YidC upon binding to the Pf3 coat substrate. Analysis of the binding curves taken from the fluorescence data yielded values for the dissociation constant (K(D)) in the range of 0.5-1.8 microM. Titration experiments with two Trp mutants reveal that the change in the tertiary structure involves mainly the membrane-spanning domain. The influence of the different environments on the secondary structure of YidC as well as of the YidC large periplasmic domain (P1) was investigated by circular dichroism (CD). The CD data show that the YidC secondary structure changes upon reconstitution into a membrane environment when compared to the detergent-solubilized state. In particular, the P1 domain of YidC is considerably affected by the detergent C(12)PC. This underlines the importance to study conformational changes with membrane-inserted proteins.
Winterfeld
Sophie
S
Institute of Microbiology, University of Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany.
Imhof
Nora
N
Roos
Tilmann
T
Bär
Gerda
G
Kuhn
Andreas
A
Gerken
Uwe
U
D002462:Cell Membrane / Q000201:enzymology*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D050505:Mutant Proteins / Q000737:chemistry; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry; D014364:Tryptophan / Q000378:metabolism
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C412443:YIDC protein, E coli; D014364:Tryptophan