title: Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.
abstract: Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (chi(1)=60 degrees and 180 degrees ) rotamers, based on the recent lifetime-rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828-35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.
authors: Unité de Chimie Organique, Institut Pasteur, URA CNRS 487, 28 rue du Dr. Roux, 75724 Paris Cedex, France.
authors: Yves-Marie
authors: YM
authors: Coïc
authors: Michel
authors: M
authors: Vincent
authors: Jacques
authors: J
authors: Gallay
authors: Françoise
authors: F
authors: Baleux
authors: Florence
authors: F
authors: Mousson
authors: Veronica
authors: V
authors: Beswick
authors: Jean-Michel
authors: JM
authors: Neumann
authors: Béatrice
authors: B
authors: de Foresta
mesh_terms: D024322:Amino Acids, Aromatic / Q000737:chemistry*; D016880:Anisotropy; D032701:Biomimetics / Q000379:methods*; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008823:Micelles; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology
chemical_list: D024322:Amino Acids, Aromatic; D005960:Glucosides; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D006180:Proton-Translocating ATPases