We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a alpha-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per microm(2) of membrane surface), the diffusion coefficient D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 microm(-2)), the lateral diffusion coefficient of the molecules decreased linearly with increasing the protein concentration in the membrane. The implications of our findings for protein mobility in biological membranes (protein crowding of approximately 25,000 microm(-2)) and use of diffusion measurements for protein geometry (size, oligomerization) determinations are discussed.

Cell membranes are comprised of multicomponent lipid and protein mixtures that exhibit a complex partitioning behavior. Regions of structural and compositional heterogeneity play a major role in the sorting and self-assembly of proteins, and their clustering into higher-order oligomers. Here, we use computer simulations and optical microscopy to study the sorting of transmembrane helices into the liquid-disordered domains of phase-separated model membranes, irrespective of peptide-lipid hydrophobic mismatch. Free energy calculations show that the enthalpic contribution due to the packing of the lipids drives the lateral sorting of the helices. Hydrophobic mismatch regulates the clustering into either small dynamic or large static aggregates. These results reveal important molecular driving forces for the lateral organization and self-assembly of transmembrane helices in heterogeneous model membranes, with implications for the formation of functional protein complexes in real cells.fluorescence microscopy | lipid rafts | membrane proteins | molecular dynamics | linactants T he heterogeneity of biological membranes plays an important role in cellular function (1, 2). Despite experimental progress in recent years (3), the characterization of lateral organization in biological membranes, however, remains challenging due to the lack of tools to study fluctuating nanoscale assemblies in living cells (4-6). Model membranes (7-10) as well as isolated plasma membranes (11, 12) are more frequently studied, because largescale phase separation can be observed in these systems. In particular, at cholesterol concentrations reminiscent of biological membranes (10-30 mol % cholesterol), ternary mixtures of saturated and unsaturated lipids can segregate into coexisting lipid domains of different fluidity, a liquid-ordered (Lo) and a liquiddisordered (Ld) phase. Probing the structural and dynamical properties of these fluid domains has received a lot of attention, as it is presumably linked to the formation of lipid nanodomains ("rafts") in real cells (13,14).The structure and function of membrane proteins is intimately connected with their lipid environment (15, 16). Because of the heterogeneity of the cell membrane, proteins partition between different lipid domains, are recruited to specific locations, and form functional complexes (17)(18)(19). This lateral organization is very important for various cellular processes, such as membrane fusion (20, 21), protein trafficking (22), and signal transduction (23-25). Although lipids and integral membrane proteins are well studied by themselves, the molecular properties that determine the specific interactions between them remain poorly understood. Interactions important for protein sorting and self-assembly are (specific) protein-protein and protein-lipid forces, and indirect lipid-mediated forces. The latter category includes, for instance, forces due to entropic confinement of lipid chains and forces arising from the mismatch between the hydrophobic parts of the protein and the bi...

In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function.

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