FRET-GP - A Local Measure of the Impact of Transmembrane Peptide on Lipids
Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of the protein with lipids. In this work we describe the FRET-GP method, which utilizes Forster resonance energy transfer (FRET) to specifically probe nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe, is quantified using generalized polarization (GP) to reports on lipid fluidity in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contribute to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters, but the local effects were considerably stronger (GPFRET >> GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby increasing comprehensibility of the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/554931v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d8fd57org.highwire.dtl.DTLVardef@e0713eorg.highwire.dtl.DTLVardef@12ab78aorg.highwire.dtl.DTLVardef@177dc30_HPS_FORMAT_FIGEXP M_FIG C_FIG