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Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.

Many proteins are known to have strong binding preferences for curved lipid bilayer surfaces. This property of proteins to sense membrane curvature has a number of implications in cellular processes including endocytosis, exocytosis, and vesicle trafficking as well as pathogenic processes associated with viral infections or protein aggregation disorders. Here we employ small unilamellar vesicles (SUVs) that are sized by extrusion through nanoporous filters as a model of convex membranes with accessible outer surface. For concave structures we developed nanopore-confined tubular lipid bilayers with tunable curvature radii ranging from ca. 40 to 250 nm. The nanotubular bilayers are formed by lipid self-assembly inside cylindrical nanopores of anodic aluminum oxide (AAO). These systems impose essentially no restrictions on the lipid composition and have the inner surface of negative curvature accessible for solute molecules and peptide binding. Using these model structures we show that the surface electrostatic potential of lipid bilayers is affected by the curvature to a rather large degree. For example, for SUVs composed of negatively charged lipids the magnitude of the surface potential increased with bilayer bending from ca. −106 mV for 100 nm SUV to −166 mV for 30 nm SUVs. These measurements were carried out by spin probe EPR method using recently synthesized lipids having pH-reporting nitroxides covalently tethered to the lipid polar head. EPR titration experiments were followed by the measurement of the lipid vesicle electrophoretic mobility and peptide binding. Overall, the data indicate that the bilayer bending affects the local electrostatic potential in a rather large degree providing a likely biophysical mechanism for affecting protein binding to lipid membranes without adjusting lipid composition. Supported by U.S. DOE Contract DE-FG02-02ER15354.

Spontaneous lipid bilayer bending and related curvature are being recognized as an essential mechanism associated with many cellular functions. Experimental observations indicate that tubular vesicles of just 50 to 100 nm in diameter are commonly formed within the Golgi body and also between the endoplasmic reticulum and the Golgi. While changes in various conditions may promote the tubulation of the Golgi membrane, detailed understanding of basic biophysical processes beyond the bilayer bending are missing in the literature and so are convenient models of highly curved lipid tubules. Here we report a systematic study of a surface potential for both small unilamellar monodisperse lipid vesicles (SUMV) with diameters ranging from 30 to 100 nm and also lipid tubules that are stabilized by confining these structures within rigid homogeneous nanopores of similar diameters. For example, for SUMVs composed of negatively charged lipids the magnitude of the surface potential increased with bilayer bending from ca. −106 mV for 100 nm SUMV to −166 mV for 30 nm SUMV. These measurements were carried out by spin probe EPR method using recently synthesized lipids having pH-reporting nitroxides covalently tethered to the lipid polar head. Parallel differential scanning calorimetry experiments indicated the presence of at least two components within the lipid phase. These phase components were characterized by measurably different phase transition temperatures and correlation times of the lipid thermal relaxation. Overall, the data indicate that the bilayer bending affects the local electrostatic potential and lipid fluctuation properties in a rather large degree and is likely associated with a mechanism for cellular machinery function. Supported by U.S. DOE Contract DE-FG02-02ER15354.

Folding of proteins with large transmembrane domains such as cellulose synthase CesA occurs in a highly heterogeneous environment of cellular membranes. Currently, detailed biophysical understanding of these processes is missing with only few examples of membrane proteins allowing for reversible folding found in the literature. Recently, in the course of ongoing investigation of the transmembrane structure of selected CesA helices, we have determined that both transmembrane helices (TMH) 4 and 5 would not insert properly into lipid bilayers and form aggregates unless specific requirements for lipid composition and/or chaperones are satisfied. In cellular membranes the roles of folding chaperons could be played by other proteins or even other domains of the same CesA. Here we have shown that an alanine-rich and generally non-aggregating membrane-spanning α-helical WALP peptide could also play such a role: when co-inserted with CesA TMHs, WALP promotes formation of transmembrane α-helices and prevents TMHs aggregation. Further, from screening of various lipids we determined that a short lipid DLPC (1,2-dilauroyl-sn-glycero- 3-phosphocholine) is the most suitable for formation of unaggregated CesA TMHs. Interestingly, a longer DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) lipid, when doped with 20 mol% of DLPC, showed even lower fraction of unaggregated peptide than in pure DLPC. Additional data on the hydrophobic matching of TMH4 and 5 to the bilayer thickness were provided by spin-labeling EPR studies. EPR data also showed that TMH4 and 5 are correctly assembled together in lipid bilayers even in the absence of the extracellular loop. Overall, the presence of various lipids in cellular membranes appears to be an important requirement for proper folding of complex integral membrane proteins. Supported as a part of the Center for LignoCellulose Structure and Formation under DOE Award DE-SC0001090.

Substrate-supported lipid bilayers serve many purposes: from acting as versatile models of cellular membranes to biotechnological applications including substrate functionalization and stabilizing membrane proteins in functional conformations. While adsorption and subsequent reorganization of phospholipid vesicles on solid substrates were studied in the past, the exact nature of physicochemical interactions between the lipids and substrate surfaces remain largely unknown. Here we employed recently synthesized pH-sensitive spin-labeled phospholipids - derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE) with pH-reporting nitroxides that are covalently attached to the lipid's headgroup - to investigate surface electrostatics of nanotubular lipid bilayers confined in cylindrical nanopores. The lipid nanotubes were formed by self-assembling phospholipids inside ordered nanochannels of anodic aluminum oxide with pore diameters from 60 to 170 nm and diameter-to-pore length ratio of up to 1:1000. 31P NMR confirmed formation of macroscopically aligned lipid nanotubes with just 1-2° mosaic spread from zwitterionic DMPC, anionic DMPG, and their mixtures. Interfacial potentials were measured by carrying out titration experiments and observing the protonation state of the nitroxide tag by EPR. For nanopore-confined DMPC:DMPG (1:1) bilayers the protonation equilibrium was shifted to more acidic values: when the single lipid bilayer was deposited per nanopore the pKa of the nitroxide probe was shifted by (−0.91±0.05) pH units but only by (−0.34±0.05) when three bilayers per nanopore were present. Notably, the nitrogen hyperfine coupling constant for non-protonated nitroxides remained the same in all the samples indicating essentially the same interfacial dielectric environment. Thus, these shifts in pKa must come from changes in the lipid bialyer surface potential that was estimated to increase by 52±3 mV. EPR data on the lipid-substrate interface were combined with differential scanning calorimetry to elucidate effects of pore curvature, surface modification, and binding of antibacterial peptides on lipid-substrate interactions. Supported by DE-FG02-02ER15354.

A growing body of experimental data suggests that at least some of membrane-anchored and membrane-associated proteins are capable of sensing the membrane curvature. Further, highly curved lipid bilayers and small vesicles are involved in such important cellular processes as membrane fusion, endo- and exocytosis, and tubules' formation. Finally, Golgi apparatus represents an example of highly curved lipid structure. While significance of membrane curvature in cellular regulatory processes is emerging, limited data exist on biophysical properties of highly curved lipid bilayers. Here we summarize results of differential scanning calorimetry and spin labeling EPR studies of unilamellar vesicles (SUV) with average diameter ranging from 200 to 30 nm. Analysis of DSC data at multiple scan rates revealed broadening and shifts of the main phase transition of DMPC from ca. 22.9 to 23.6 oC. This observation is consistent with bilayer compression and an increase in local order parameter reveled by EPR and oxygen accessibility measurements. To assess the surface electrostatics of lipid vesicles we employed EPR of a recently introduced phospholipid (IMTSL-PTE) bearing a pH-sensitive nitroxide covalently attached to the lipid head group (Biophys. J. 2013, 104: 106). The magnitude of the negative surface electrostatic potential, Ψ, for POPG increased from −137 to −167 mV upon decrease in the vesicle diameter from 107 to 31 nm even though zeta-potentials were identical. This effect could be again rationalized by increase in lipid packing upon increase in curvature for the bilayer in fluid phase. However, the effect vanished for the gel phase. We conclude that biologically relevant fluid bilayer phase allows for a larger variability in the lipid packing density in the lipid polar head group region than a more ordered gel phase. Supported by U.S. DOE Contract DE-FG02-02ER15354.