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Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues

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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 (λmax), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (kq) 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 (χ1=60° and 180°) 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.

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Abbreviations

PMP1 (or 2):

Plasma membrane proteolipid 1 (or 2)

PLN:

Phospholamban

SLN:

Sarcolipin

DM:

Dodecylmaltoside

BrDM:

7,8-dibromododecylmaltoside

BrUM:

10,11-dibromoundecanoylmaltoside

DPC:

Dodecylphosphocholine

SDS:

Sodium dodecyl sulfate

POPS:

1-palmitoyl-2-oleoyl-3-glycerophosphatidylserine

NATA:

N-acetyltryptophanamide

TOE:

Tryptophan octyl ester

SR:

Sarcoplasmic reticulum

MEM:

Maximum entropy method

P3:

K2WL9AL9K2A

P5:

K2CLWL7AL9K2A

P7:

K2CL3WL5AL9K2A

P9:

K2CL5WL3AL9K2A

P11:

K2CL7WLAL9K2A

P13:

K2CL9WL9K2A

HATU:

O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

DIPEA:

N,N-diisopropylethylamine

TFA:

Trifluoroacetic acid

MPLC:

Medium-pressure liquid chromatography

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Acknowledgements

We thank the technical staff of the Laboratoire pour l’Utilisation du Rayonnement Electromagnétique (LURE) for running the synchrotron ring during the beam sessions.

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Author notes

  1. Michel Vincent & Jacques Gallay

    Present address: Centre Universitaire Paris-Sud, UMR CNRS 8619, Bât. 430, 91405, Orsay Cedex, France

  2. Veronica Beswick

    Present address: Université d’Evry, Bd F. Mitterrand, 91025, Evry, France

  3. Florence Mousson

    Present address: Laboratory for Physiological Chemistry, University Medical Centre-Utrecht, 3508, Utrecht, AB, The Netherlands

Authors and Affiliations

  1. Unité de Chimie Organique, Institut Pasteur, URA CNRS 487, 28 rue du Dr. Roux, 75724, Paris Cedex, France

    Yves-Marie Coïc & Françoise Baleux

  2. Laboratoire pour l’Utilisation du Rayonnement Electromagnétique, Université Paris-Sud, UMR CNRS 130, Orsay, France

    Michel Vincent & Jacques Gallay

  3. Service de Biophysique des Fonctions Membranaires, Centre d’Etudes de Saclay, CEA DSV/DBJC and URA CNRS 2096, 91191, Gif sur Yvette Cedex, France

    Florence Mousson, Veronica Beswick, Jean-Michel Neumann & Béatrice de Foresta

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  1. Yves-Marie Coïc
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Correspondence to Béatrice de Foresta.

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Coïc, YM., Vincent, M., Gallay, J. et al. Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues. Eur Biophys J 35, 27–39 (2005). https://doi.org/10.1007/s00249-005-0002-1

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