YidC-driven membrane insertion of single fluorescent Pf3 coat proteins.

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.

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.

Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible.

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.

Proton-decoupled 15N and 31P solid-state NMR investigations of the Pf3 coat protein in oriented phospholipid bilayers.

The coat proteins of filamentous phage are first synthesized as transmembrane proteins and then assembled onto the extruding viral particles. We investigated the transmembrane conformation of the Pseudomonas aeruginosa Pf3 phage coat protein using proton-decoupled 15N and 31P solid-state NMR spectroscopy. The protein was either biochemically purified and uniformly labelled with 15N or synthesized chemically and labelled at specific sites. The proteins were then reconstituted into oriented phospholipid bilayers and the resulting samples analysed. The data suggest a model in which the protein adopts a tilted helix with an angle of approximately 30 degrees and an N-terminal 'swinging arm' at the membrane surface.