Lipid-protein interaction

Together with lipids, proteins are the main constituents of natural membranes, and are implicated in all biochemical processes occurring in the membrane. Membrane proteins include integral proteins that are embedded in the bilayer or span the membrane, and peripheral proteins that are bound to the membrane surface. Both these types of proteins are studied in our lab. Whereas the knowledge of the phase behavior of multicomponent lipid systems is the starting point, the understanding of biomembranes requires knowledge about the molecular interactions of all components, notably lipid-protein interactions.

Relevant problems that are addressed in our group using fluorescence methodologies are:

1. Quantification of the extent of interaction of the peptide/protein with the membrane (fluorescence intensity/lifetime/anisotropy, Figure 1);

   Figure 1 - Variation of the lifetime-weighted quantum yield of α-MSH ( λexc = 295 nm) vs. lipid concentration [L] of DMPC/DMPG (3:1) (A and B), and DMPC/DMPA (3:1) (C and D) in the gel phase at 20ºC (A and C) and in the fluid phase at 37ºC (B and D), as increasing amounts of peptide partition to the bilayers. Peptide concentration (~30 μM) was kept constant during the experiments. The solid line is the fit of the partition model to the data.

2. Determining the transverse location of the peptide/protein fluorophore (quenching, FRET, spectral changes);
3. Obtaining information on the secondary structure (Figure 2)/dynamics of the membrane-bound peptide/protein (fluorescence intensity and anisotropy decays);

   Figure 2 - Model of the β-hairpin proposed for the ShB peptide inserted into palmitoyloleoylphosphatidic acid (POPA) vesicles. Phospholipids in the bilayer are colored in yellow. The peptide surface is colored according to the electrostatic potential (blue, electropositive, and red, electronegative charges). The β-structure backbone and Tyr-8 residue are colored in green. The dotted lines represent the bilayer surface.

4. Formation of protein/peptide-rich patches or protein/peptide aggregates vs. random distribution (fluorescence self-quenching, FRET (both homo- and heterotransfer, Figure 3));

   Figure 3 - Putative organization of γM4 in Chol-poor and Chol-rich systems. A plausible structural framework to account for the absence of energy migration between Trp453 residues in the γM4 peptide, and the variation of lifetime-weighted quantum yield with peptide concentration on the ld and lo phases is provided. A) Top view of the α-helical peptide showing the relative positions of Trp453 and Cys451, and a probable geometry for a disulfide bonded dimer. B) Possible geometry for a linear aggregate. This structure is ruled out. C) Parallel aggregate in end-on view. This structure is not ruled out by energy homotransfer data. D) Anti-parallel aggregate, lateral view. This structure is not ruled out by energy homotransfer data. E) Peptide-rich patch. The model depicts the distribution of γM4 in POPC/Chol vesicles with high Chol content (lo phase). The negative mismatch causes a disordering effect in the vicinity of a peptide molecule, and other peptides accommodate better in a region close to where other peptides localize. In the ld (Chol-poor phase) the peptide is randomly distributed due to the very good matching between the hydrophobic thickness of the bilayer and the peptide.

5. Formation of protein/peptide-rich patches or protein/peptide aggregates vs. random distribution (fluorescence self-quenching, FRET (both homo- and heterotransfer, Figure 3));

   Figure 4 - Schematic representation of M13 major coat protein in a bilayer, together with the FRET model derived for characterization of the selectivity of this protein for different lipid species.

6. Peptide-mediated vesicle fusion/aggregation (FRET, Figure 5).

   Figure 5 - Schematic representation of the action of cationic peptide K6W upon mixtures of DPPC (zwitterionic lipid with white headgroup in the cartoon) and DPPS (anionic lipid with orange headgroup in the cartoon). The formation of a multibilayer structure was unveiled by global analysis of time-resolved FRET data.

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