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.

Structural Dynamics of the YidC:Ribosome Complex during Membrane Protein Biogenesis

“…Interestingly, similar membrane relocating had been recently described for the amphipathic helix of the TatA subunit of the twin-arginine translocon, where the biophysical analysis in model membranes has been supported by an NMR-based structure (Chan et al., 2011, Rodriguez et al., 2013). Although EH1 of YidC has been suggested to reside at the membrane interface upon post-translational insertion (Imhof et al., 2011), the dynamics of YidC upon ribosome binding and co-translational insertion observed here is clearly different.…”

Dynamic Disulfide Scanning of the Membrane-inserting Pf3 Coat Protein Reveals Multiple YidC Substrate Contacts

“…During this process, the Pf3 protein binds to YidC mainly by hydrophobic interactions (9) and induces a conformational change within YidC (10,25). As a result, the Pf3 coat protein adopts a transmembrane conformation, exposing its N-terminal domain to the periplasm.…”

Fluorescence in Nanobiotechnology: Sophisticated Fluorophores for Novel Applications

“…Lamp Halogen: broad spectrum (UV/vis), steady-state emission, very inexpensive Spectrofl uorometers, microplate readers, time-resolved applications, detection and genotyping of mRNA [ 49 ] Xe: broad spectrum (UV/vis), high output power, steady-state and pulsed up to hundreds of Hz Hg and Hg-Ar: distinct lines with high power, Vis and IR LED (light-emitting diodes) Continuous wave (cw) or pulsed output up to MHz frequencies, spectral broadness about 20 nm, UV and Vis, usually low to moderate output power Spectrofl uorometers, excitation sources for time-resolved techniques, investigation of conformation dynamics of an E. coli membrane insertase [235] Laser (light amplifi cation by stimulated emission of radiation)…”