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Single molecule diffusion of membrane-bound proteins: window into lipid contacts and bilayer dynamics.

ABSTRACT: Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids--an insight with significant implications for theoretical description of bilayer lipid dynamics.

SUBMITTER: Knight JD

PROVIDER: S-EPMC2966005 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Single molecule diffusion of membrane-bound proteins: window into lipid contacts and bilayer dynamics.

Knight Jefferson D JD Lerner Michael G MG Marcano-Velázquez Joan G JG Pastor Richard W RW Falke Joseph J JJ

Biophysical journal 20101101 9

Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. ...[more]

PMID: 21044585

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Project description:Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1-3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both (1) individual bound lipids and (2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2D diffusion constant. An empirical formula is developed that accurately estimates the diffusion constant and bilayer friction of a peripheral protein in terms of its number of bound lipids and its geometry of penetration into the bilayer hydrocarbon core, yielding an excellent global best fit (R(2) of 0.97) to the experimental diffusion constants. Finally, the observed additivity of the frictional contributions suggests that further development of current theory describing bilayer dynamics may be needed. The present findings provide constraints that will be useful in such theory development.

Project description:Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus, a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca(2+)-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves, identifies and quantitates the rapidly diffusing monomers and slowly diffusing heterodimers. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing ratio of monomers and heterodimers, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ~10(2)-fold greater in the membrane system than in solution, apparently due to both faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.

Project description:The human voltage dependent anion channel 1 (VDAC) is a 32 kDa ?-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane ?-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

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Single molecule diffusion of membrane-bound proteins: window into lipid contacts and bilayer dynamics.

Publications

Single molecule diffusion of membrane-bound proteins: window into lipid contacts and bilayer dynamics.

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