Flexible Surface Model Research Articles

Packing and elasticity in membrane mechanical properties.

Here we address how membrane properties involving head group size, acyl chain unsaturation, and lipid packing affect their structure and dynamics. Strong out-of-plane proteolipid couplings considered by the flexible surface model (FSM) affect protein function through elastic forces described by the spontaneous curvature and the bending rigidity. We hypothesized that the mechanical properties and the molecular packing resulting from different lipid types are captured by introducing a perdeuterated POPC-d31 probe lipid. Di-monounsaturated phosphatidylcholine and phosphatidylethanolamine (DOPC, DOPE), and different ratios of DOPC/DOPE mixtures were studied using MD simulations combined with experimental solid-state 2H NMR spectroscopy. Residual quadrupolar couplings obtained from de-Paked powder-type spectra yielded order parameter (SCD) profiles for the individual acyl segments giving insights into average membrane structural properties such as area per lipid and bilayer thickness [1]. The corresponding spin-lattice relaxation rates (R1Z) for the resolved peaks followed a theoretical square-law dependence on SCD providing the bending rigidity [2]. Furthermore, MD simulations for the lipid systems were carried out and order parameters were calculated and compared against the NMR-observed SCD values. Utilization of the probe lipid POPC-d31 (1−10 mol%)affected the lamellar to hexagonal phase transition temperature of DOPE lipid systems but did not affect the quadrupolar splittings of DOPE and DOPC systems. Higher bending rigidity was observed for DOPE membranes versus DOPC supporting the idea that the membrane bending rigidity is primarily governed by increased lipid packing and is related to the area-compressibility modulus. Use of a probe lipid to capture membrane mechanical properties of complex lipid systems allow investigation of the relationship of membrane mechanics in vital biological functions.

Membrane Curvature Revisited—the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.

The spherical droplet model extended: The relation between surfactant packing and aggregate composition

The classical emulsion droplet models found in the literature are based on a Volume/Area ratio, which gives the minimum droplet size of a monodisperse system at a fixed composition. These models have been revised and summarized into a general equation. Additionally, an alternative model based on a Volume/Volume ratio has been introduced which shows a non-linear behavior between droplet size and composition. At the micellar domain, the introduced droplet model shows a different behavior from the classical approach and also when combined with the flexible surface model or with the Israelachvili packing equation. To understand the differences between models, the packing of surfactant molecules at the droplet interface has been studied as a function of composition. A spherical packing parameter (Pagg) has been defined which, as a difference with Israelachvili´s concept, includes the penetration of oil and water molecules into the surfactant palisade layer. It has been described a semi-empirical method to estimate Pagg in monodisperse droplet like systems from simple size measurements. On the other hand, an equation showing the relation between Pagg and droplet composition has been deduced and applied on O/W discrete cubic liquid crystalline phases, microemulsion and nano-emulsion systems formed in selected Water / Polyethoxylated alcohol / Oil systems.

Photocatalytic Cleavage of C–C Bond in Lignin Models under Visible Light on Mesoporous Graphitic Carbon Nitride through π–π Stacking Interaction

Photocatalysis is a potentially promising approach to harvest aromatic compounds from lignin. However, the development of an active and selective solid photocatalyst is still challenging for lignin transformation under ambient conditions. We herein report a mild photocatalytic oxidative strategy for C–C bond cleavage of lignin β-O-4 and β-1 linkages using a mesoporous graphitic carbon nitride catalyst. Identifications by solid-state NMR techniques and density functional theory (DFT) calculations indicate that π–π stacking interactions are most likely present between the flexible carbon nitride surface and lignin model molecule. Besides, low charge recombination efficiency and high specific surface area (206.5 m2 g–1) of the catalyst also contribute to its high catalytic activity. Mechanistic investigations reveal that photogenerated holes, as the main active species, trigger the oxidation and C–C bond cleavage of lignin models. This study sheds light on the interaction between complex lignin structures an...

Emergence of Membrane Material Parameters Revealed by Solid-State 2H NMR Spectroscopy

The emergence of the material properties of lipid membranes is a topic of current interest in quantitative biophysics. At the smallest length scale, lipid membranes are collections of atoms whose dynamics can be traced by molecular dynamics simulations. At cellular length scales, they are continuous hydrophobic barriers, which together with membrane proteins regulate trafficking and cell signaling. Somewhere in this broad range, material properties emerge from the ensemble of atoms. How can we understand this emergence behavior? Here we show that solid-state 2H NMR spectroscopy is powerful, because as opposed to typical scattering experiments it provides information over a range of length scales. Specifically, solid-state 2H NMR provides atomistically resolved order parameters and dynamics of flexible phospholipids within the liquid-crystalline bilayer [1]. By measuring the membrane response to osmotic stress we can disentangle how bulk material properties emerge from atomistic-level interactions. Examples include structural parameters such as the area per lipid and the volumetric thickness plus the associated moduli for elastic deformation. Data for samples under osmotic stress allow an insightful analysis of intermembrane interactions by distinguishing between collective undulatory and quasi-elastic contributions at large length scales versus short-range non-collective (molecular) effects. By calculating the area elastic modulus over a substantial range of osmotic pressures, collective bilayer properties are found to emerge over very short length scales, on the order of the bilayer thickness or even less. Notably the emergence of lipid material properties on this length scale determines what model is most appropriate to study lipid-protein interactions [2]. A continuum flexible surface model (FSM) for lipid-protein interactions on the mesoscopic length scale can optimally capture the essential material features that govern biomembrane functions. [1] J.Kinnun et al. (2015) Biochim.Biophys.Acta 1848, 256. [2] M.F.Brown (2017) Annu.Rev.Biophys. 46, 379.

Soft Matter in Lipid-Protein Interactions.

Membrane lipids and cellular water (soft matter) are becoming increasingly recognized as key determinants of protein structure and function. Their influences can be ascribed to modulation of the bilayer properties or to specific binding and allosteric regulation of protein activity. In this review, we first consider hydrophobic matching of the intramembranous proteolipid boundary to explain the conformational changes and oligomeric states of proteins within the bilayer. Alternatively, membranes can be viewed as complex fluids, whose properties are linked to key biological functions. Critical behavior and nonideal mixing of the lipids have been proposed to explain how raft-like microstructures involving cholesterol affect membrane protein activity. Furthermore, the persistence length for lipid-protein interactions suggests the curvature force field of the membrane comes into play. A flexible surface model describes how curvature and hydrophobic forces lead to the emergence of new protein functional states within the membrane lipid bilayer.

Computational and Experimental Studies of Lipid-Protein Interactions in Biomemrane Function

Integral membrane proteins constitute more than 30% of the human genome. Substantial lipid-protein interactions contribute to the structure and function of membrane proteins such as GPCRs and viral membrane ion channels. We address how physical properties of the membrane as well as positive charge on the lipid head group modulate protein activation. The flexible surface model (FSM) describes physical properties of the lipid environment that effect membrane protein activation [3]. According to the FSM, elastic coupling of the membrane lipids to integral membrane proteins define lipid-protein interactions through a balance of curvature and hydrophobic forces. Using UV-visible spectroscopy, FTIR spectroscopy [2], and molecular dynamics (MD) simulations, we discovered that lipid bilayer properties like head group and chain length modulate membrane protein functioning by influencing the conformational states. Using rhodopsin, a canonical G-protein-coupled receptor (GPCR) as a prototype, we show the effect of positively charged membrane lipid head group on rhodopsin activation using UV-visible spectroscopy and MD simulations. We discovered rhodopsin reconstituted in membrane lipids with a positively charged head group like DOTAP shifts the equilibrium to active Meta-II state. We propose a mechanism where membrane lipids with positively charged head group interact with Glu134 present on lipid-protein interface breaking the salt bridge with Arg135 in the conserved ERY motif. The interaction between Glu134 and the positively charged head group stabilizes the active Meta-II state. Our study gives insight on the role of membrane lipid-protein interactions in rhodopsin activation mechanism which can ultimately be extended to influenza virus membrane ion channels and other integral proteins. [1] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [2] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [3] M.F. Brown (2012) Biochemistry 51, 9782-9795.

Membrane-Lipid Mediated Rhodopsin Signaling Involves an Ensemble of Conformational Substates

G-protein-coupled receptors (GPCRs) constitute about 50% of the known drug targets. Using rhodopsin as a model GPCR, we study the role of membrane lipids in the rhodopsin activation. Absorption of light by rhodopsin leads to a series of conformational changes that establishes an equilibrium between inactive Meta-I and an ensemble of activated Meta-II states [1,2]. Using UV-visible and FTIR spectroscopy, we show that the distribution of late conformational substates during rhodopsin activation is lipid-mediated as explained by an ensemble activation mechanism (EAM). Lipid bilayer composition (head groups and acyl chain lengths) and membrane protein-lipid interaction govern the EAM through biasing conformational substates. Rhodopsin reconstituted in DOPC backshifts the equilibrium to the Meta-IIa substate, whereas mixed-chain POPC membranes favor the inactive Meta-I state [3]. The wavenumber-dependent analysis of the FTIR-difference spectra yields a distribution of pKa and alkaline endpoint values, consistent with an ensemble of substates for each lipid bilayer-rhodopsin system. A phenomenological Henderson-Hasselbalch function was fitted to the pH titration curves to derive thermodynamic parameters from the spectral analysis. Our thermodynamic studies show that activation of rhodopsin is accompanied by an entropy gain compensating the unfavorable enthalpy increase, analogous to protein unfolding reactions. The results from the EAM analysis are in agreement with the flexible surface model (FSM), which describes elastic coupling of the membrane lipids to integral membrane proteins through a balance of curvature and hydrophobic forces in lipid-protein interactions [4]. Our study also provides insight into the thermodynamic parameters that govern rhodopsin-like GPCR activation in native membrane lipid environments. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A.V. Struts et al. (2014) Meth. Mol. Biol. (in press). [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [4] M.F. Brown (2012) Biochemistry 51, 9782-9795.

Investigation of Lipid Bilayer Effects on Rhodopsin Activation using UV-Visible and Ftir Spectroscopy

G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets and play crucial roles in signal transduction for a number of physiological processes. Upon photoactivation, rhodopsin undergoes a series of conformational changes leading to visual perception. An ensemble of activated Meta II states is in equilibrium with the inactive precursor Meta I [1]. Lipid bilayer composition and its interaction with membrane proteins govern the ensemble activation mechanism (EAM) as predicted by the flexible surface model (FSM). The FSM describes the balance between intrinsic monolayer curvature and lipid-protein hydrophobic interactions, leading to the elastic coupling of lipids and membrane proteins [2]. Effects of temperature and pH were analyzed for rhodopsin reconstituted in lipid vesicles using UV-visible and FTIR spectroscopy. Thermodynamic properties were derived by fitting phenomenological Henderson-Hasselbalch functions to their respective pH titration curves. Mixed-chain POPC membranes backshift rhodopsin towards Meta I, whereas rhodopsin in DOPC favors the active Meta IIa substate. Analysis of the wavelength-dependent distribution of pKa and alkaline endpoints as estimated from FTIR difference spectra reveals an ensemble of substates for each lipid bilayer-rhodopsin system. The presence of multiple activated conformations is a hallmark of the EAM. Our results are in agreement with the FSM, whereby lipids having a negative monolayer curvature favor the active Meta II state, while lipids with zero spontaneous curvature (POPC) favor the inactive Meta I state [3]. The data give additional insight into the entropy-enthalpy balance which drives the structural conformational changes that occur upon rhodopsin photoactivation. Moreover our work provides fundamental insight into the functionality of other GPCR-related proteins in a natural membrane lipid environment. [1] A.V. Struts (2011) PNAS 108, 8263-8268. [2] M.F. Brown (2012) Biochemistry 51, 9782-9795. [3] E. Zaitseva (2010) JACS 132, 4815-4821.

Role of Membrane Lipids in Activating G-Protein-Coupled Receptors

The role of lipid-protein interactions in membrane function has long attracted the attention of researchers in the field of lipid membrane biophysics. Effects of membrane lipids on G-protein-coupled receptors (GPCRs) are revealed by UV-visible and FTIR spectroscopic studies of the conformational energetics of rhodopsin in visual signaling [1]. During rhodopsin photoactivation, the photoreactive 11-cis-retinylidene chromophore is isomerized to all-trans yielding an equilibrium between inactive Meta-I and active Meta-II states. Modulation of the metarhodopsin equilibrium depends on the polar head groups and the lipid acyl chain length and polyunsaturation. Membrane lipids can forward or back-shift the metarhodopsin equilibrium due to their chemically non-specific material properties [2]. A flexible surface model (FSM) describes elastic coupling of membrane lipids to the conformational energetics of rhodopsin. The new biomembrane model challenges the standard fluid mosaic model. Based on data first introduced for rhodopsin [2] the idea of a curvature stress field bridges theory and experiment. According to the FSM, membrane lipids whose spontaneous curvature stabilizes the activated state within the membrane are involved in regulating protein function. The new biomembrane model explains the effects of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on visual signaling. An ensemble-mediated activation mechanism is proposed for rhodopsin in a natural membrane lipid environment, which includes a role for bulk water in the activation of rhodopsin-like GPCRs [4]. Ion channels, transporters, and membrane-bound peptides can all be affected by curvature forces due to elastic deformation of the bilayer, thus giving a new paradigm for membrane lipid-protein interactions in structural biology.[1] M.F. Brown (2012) Meth. Mol. Biol.914, 127-153.[2] M.F. Brown (1997) Curr. Top. Membr.44, 285-356.[3] M.F. Brown (2012) Biochemistry51, 9782-9795.[4] A.V. Struts (2011) PNAS 108, 8263-8268.

Membrane Bilayer Environment Influences Thermodynamics of Rhodopsin Membrane Protein-Lipid Interactions

The interaction between lipid bilayers and membrane proteins plays an important role in the functionality of GPCRs such as rhodopsin. During rhodopsin photoactivation, the photoreactive 11-cis retinylidene chromophore is isomerized to an all-trans state attaining an equilibrium between the MetaI and MetaII states. Lipid bilayer interaction with rhodopsin during its photoactivation process is explained using the flexible surface model (FSM). The FSM describes the elastic coupling of membrane lipids to integral membrane proteins through a balance of curvature and hydrophobic forces in lipid-protein interactions [1]. The FSM predicts a shift of the MetaI-MetaII equilibrium towards the activated MetaII state for unsaturated phosphatidylcholines with an increase in acyl chain length [1] or temperature. using UV-visible spectroscopy, we test the FSM by evaluating how the MetaI-MetaII equilibrium and thermodynamic parameters are influenced by a change in membrane environment. Absorption wavelength maxima at 485 nm and 380 nm correspond to the MetaI and MetaII states (inactive and active MetaII-H+), respectively. Accurate results are obtained by using the Rayleigh-Gans approximation to Mie scattering to compensate for light scattering in the UV-visible spectra. We determine effects of the type of head group, lipid to protein ratio, acyl chain length, and temperature on the MetaI-MetaII equilibrium by comparing pH titration curves for reconstituted rhodopsin in different lipid bilayers to those for native disk membranes [2,3]. The thermodynamic parameters are obtained for this equilibrium by fitting a modified phenomenological Henderson-Hasselbalch function [2] to the pH titration curves. These thermodynamic parameters illustrate how free energy drives the structural changes in rhodopsin upon photoactivation and can be used to model other GPCRs.[1] A.V. Botelho et al. (2006) BJ91, 4464-4477.[2] M. Mahalingam et al. (2008) PNAS105, 17795-17800.[3] E. Zaitseva et al. (2010) JACS132, 4815-4821.

Lipid Bilayer Influences Rhodopsin Activation Probed by FTIR and UV-Visible Spectroscopy

Rhodopsin, the mammalian dim light photoreceptor, is the model protein for studying G protein-coupled receptor (GPCR) structure and function. We postulate that this process can be attributed to an ensemble of activated states - the ensemble activation mechanism (EAM) - and that the EAM is directly affected by membrane protein-lipid bilayer interactions dependent upon cell membrane composition, as predicted by the flexible surface model [1]. Rhodopsin was detergent-solubilized and incorporated into homogenous lipid vesicles (either POPC or DOPC) by dialysis. Samples were prepared at a series of temperature and pH values, bleached, and analyzed by simultaneous Fourier transform infrared (FTIR) and UV-visible spectral acquisition. Activation in mixed-chain POPC bilayers drastically backshifts rhodopsin from the active Meta II photointermediate to the inactive Meta I state. A di-monounsaturated phospholipid like DOPC restores partial activity to rhodopsin, as well as stabilizing the protein in the Meta IIa state. Spectral reduction and analysis via pH titration curves yielded non-Henderson-Hasselbach behavior, indicating more than one activated state exists, thus supporting the concept of an EAM [2]. In addition, temperature changes have a marked effect on rhodopsin activation, decoupling the two protonation switches necessary for full activation at physiological temperatures. By manipulating the lipid environment, we validate the flexible surface model and EAM. Lipids with negative monolayer curvature such as DOPC facilitate rhodopsin activation towards the Meta II state [3]. Thermodynamic parameters showed that free energy changes are related to greater flexibility of rhodopsin in the cell membrane upon activation. Further validation of the flexible surface model is an important contribution to biophysical understanding of GPCR function.[1] A.V. Botelho et al. (2006) BJ 91, 4464-4477. [2] M. Mahalingam et al. (2008) PNAS 105, 17795-17800. [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821.

Curvature Forces in Membrane Lipid–Protein Interactions

Membrane biochemists are becoming increasingly aware of the role of lipid-protein interactions in diverse cellular functions. This review describes how conformational changes in membrane proteins, involving folding, stability, and membrane shape transitions, potentially involve elastic remodeling of the lipid bilayer. Evidence suggests that membrane lipids affect proteins through interactions of a relatively long-range nature, extending beyond a single annulus of next-neighbor boundary lipids. It is assumed the distance scale of the forces is large compared to the molecular range of action. Application of the theory of elasticity to flexible soft surfaces derives from classical physics and explains the polymorphism of both detergents and membrane phospholipids. A flexible surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. Chemically nonspecific properties of the lipid bilayer modulate the conformational energetics of membrane proteins. The new biomembrane model challenges the standard model (the fluid mosaic model) found in biochemistry texts. The idea of a curvature force field based on data first introduced for rhodopsin gives a bridge between theory and experiment. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress are all explained by the FSM. An increased awareness of curvature forces suggests that research will accelerate as structural biology becomes more closely entwined with the physical chemistry of lipids in explaining membrane structure and function.

Flexible Surface Model for Membrane Lipid-Protein Interactions

Membrane protein conformational changes, folding and stability, and membrane fusion may all involve elastic deformation of the bilayer. The development of solid-state deuterium NMR spectroscopy [1] provides a basis for experimentally investigating lipid structure and dynamics under external forces due to temperature, osmotic pressure, or lipid composition. Deuterium NMR relaxation illuminates long-range collective lipid interactions [1]. Non-specific properties of the bilayer play a significant role in modulating protein conformational energetics [2]. A flexible-surface model (FSM) describes the balance of curvature and hydrophobic forces in lipid-protein interactions. The FSM describes elastic coupling of membrane lipids to integral membrane proteins. Curvature and hydrophobic matching to the lipid bilayer entails a stress field that explains membrane protein activity and stability [3]. Rhodopsin provides an important example, where solid-state NMR studies [4,5] and FTIR spectroscopy [6] characterize the energy landscape of the dynamically activated receptor. Upon light activation rhodopsin becomes a sensor of negative spontaneous curvature [2]. Time-resolved UV-visible and FTIR spectroscopic studies show how membrane lipids forward or back-shift the metarhodopsin equilibrium due to chemically non-specific material properties [2]. Influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on rhodopsin function are all explained by the new biomembrane model. By contrast, the older fluid-mosaic model including lipid rafts fails to account for such effects on membrane protein activity. According to the FSM, proteins are regulated by membrane lipids whose spontaneous curvature most closely matches the activated state within the lipid membrane. [1] M.F. Brown (1996) in Membrane Structure & Dynamics, pp. 175-252. [2] M.F. Brown (1994) Chem. Phys. Lipids 73, 159-180. [3] A.V. Botelho et al. (2006) Biophys. J. 91, 4464. [4] A.V. Struts et al. (2011) Nature Struct. Mol. Biol. 18, 392-394. [5] A.V. Struts et al. (2011) PNAS 20, 8263-8268. [6] M. Mahalingam et al. (2008) PNAS 105, 17795-17800.

Rhodopsin Activation is Modulated by Non-Specific Membrane Lipid-Protein Interactions

Rhodopsin activation proceeds through an ensemble of conformational substates: Meta I ↔ Meta IIa ↔ Meta IIb ↔ Meta IIbH+ [1]. These substates are characterized by Schiff base deprotonation (Meta I), an outward tilt of helix H6 (Meta IIb), and protonation of Glu134 (Meta IIbH+). Lipid bilayer composition has been shown to affect rhodopsin activation, providing an opportunity to systematically investigate membrane protein-lipid bilayer interactions on the mesoscale [2]. To quantify lipid acyl chain effects on GPCR activation, rhodopsin was reconstituted in lipids with symmetric unsaturated acyl chains (DOPC) or asymmetric lipids with sn-1 saturated and sn-2 unsaturated acyl groups (POPC). Proteolipid recombinant membranes were studied by UV-visible and FTIR spectroscopy. Symmetric membrane lipids (DOPC) stabilize Meta IIa and render the normally weakly populated substate accessible to study. The Meta IIa substate is characterized by an opening of the Schiff base ionic lock, and an activation switch in a conserved water-mediated, hydrogen-bonded network involving helices H1/H2/H7 that is sensed by Asp83. Replacement of an unsaturated acyl chain with a saturated chain (POPC) increases the pKa value for the Meta I ↔ Meta II equilibrium, and consequently destabilizes the Meta IIa substate. Modulation of the bilayer curvature stress due to a negative monolayer spontaneous curvature (H0) (DOPC) contributes a mechanical force that shifts the Meta I ↔ Meta II equilibrium towards Meta II, leading to rhodopsin activation. The flexible surface model (FSM) explains how chemically non-specific interactions between membrane proteins and the lipid bilayer contribute to GPCR activation [3,4].[1] M. Mahalingam et al. (2008) PNAS 105:17795.[2] E. Zaitseva et al. (2010) JACS 132:4815.[3] A.V. Botelho et al. (2006) Biophys. J. 91:4464.[4] M.F. Brown (1994) Chem. Phys. Lipids 73:159.

Time-Resolved UV-Visible Studies of Rhodopsin Provide Experimental Test of Flexible Surface Model for Lipid-Protein Interactions

Time-resolved UV/Visible absorbance of the retinal chromophore of rhodopsin [1] delivers vital information about key GPCR activation steps in a membrane lipid environment [2]. Following rhodopsin photoexcitation, a minimum of four intermediates equilibrate on the millisecond-time scale [2,3]. The first equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. A second equilibrium entails spectrally silent conversion of Meta IIa to the Meta IIb substate. The final step is protonation of Glu134 of the ERY sequence in Meta IIb to yield Meta IIbH+ whose pKa characterizes the acid-base equilibrium [3]. Absorbance measurements on the microsecond-to-hundred millisecond-time scale allowed us to study effects of POPC, DOPC, or DOPC/DOPE mixtures on the first equilibrium constant K1 and on the pKa of the final equilibrium. Results were analyzed by singular-value decomposition and were fit by a linear combination of temperature-dependent basis spectra. Notably an increase in K1 was discovered due to either PE head groups or increased acyl chain unsaturation, whereas little change in pKawas evident. According to the flexible-surface model (FSM) rhodopsin becomes a sensor of negative spontaneous (intrinsic) curvature upon light activation [4]. Competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary explains the influences of lipid-protein interactions [4]. Both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition, revealing how protein energetics are affected by material properties of the lipid bilayer.[1] A.V. Struts et al. (2011) Nature Struct. Mol. Biol. 18, 392-394.[2] J. Epps et al. (2006) Photochem. Photobiol. 82, 1436-1441.[3] M. Mahalingam et al. (2008) PNAS 105 17795-17800.[4] A. V. Botelho et al. (2006) Biophys. J. 91, 4464-4477.

Flexible Surface Model Explains Time-Resolved Uv-Visible Studies of Rhodopsin Activation in a Membrane Lipid Environment

Rhodopsin is the quintessential GPCR--the UV/Visible absorbance of its bound chromophore provides detailed time-resolved information about the GPCR activation steps [1]. At least four species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane environment [2]. The first millisecond-time scale equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. Subsequent equilibria involve spectrally silent transitions of Meta IIa to produce Meta IIb and protonation of Glu134 of the E(R)Y sequence in Meta IIb to give Meta IIbH+ where pKa describes the acid-base equilibrium. We used time-resolved absorbance measurements on the microsecond-to-hundred millisecond time scale to study effects of the membrane lipid environment on the first equilibrium constant, K1, and on the pKa of the final equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or DOPC/DOPE mixtures were studied at 30oC. Results were analyzed by singular value decomposition and globally fit to a sum of exponential terms. We discovered a striking increase in K1 due to either PE head groups or increased acyl chain unsaturation; by contrast pKa changed little. According to the flexible surface model (FSM), there is competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary that explains the above influences of lipid-protein interactions [3]. The fact that both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition of rhodopsin is a striking illustration of how protein energetics in membranes are governed by chemically nonspecific properties of the lipid bilayer. [1] J. Epps et al. (2006) Photochem. Photobiol.82, 1436-1441. [2] E. Zaitseva et al. (2010) JACS132, 4815-4821. [3] A.V. Botelho et al. (2006) BJ91, 4464-4477.

A Flexible Hingeless Control Surface Inspired by Aquatic Animals

A flexible hingeless control surface model was proposed for motion control of Underwater Vehicles (UVs), which is inspired by the flexible bending control surfaces of underwater creatures, such as fish and squid. Computational Fluid Dynamics (CFD) simulation demonstrates that, in comparison with the hinged or rigid control surface, the proposed flexible bending control surface can suppress the flow separation so as to improve the turning performance. A prototype of the flexible control surface was fabricated, in which Shape Memory Alloy (SMA) wires were selected as the actuators. The elastic energy storage and exchange mechanism was incorporated into the actuation of the control surface to improve the efficiency. Thermal analysis of SMA wires was performed to find proper actuating condition. Open-loop bending experiments were carried out. The results show that the proposed control surface can achieve the maximum bending angle of 104°. Moreover, the power and energy consumption under different pulse conditions were compared.

Curvature and Hydrophobic Forces Drive Oligomerization and Modulate Activity of Rhodopsin in Membranes

G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the p K a for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.

Evidence for Specificity in Lipid-Rhodopsin Interactions

The interaction of bovine rhodopsin with poly- and monounsaturated lipids was studied by (1)H MAS NMR with magnetization transfer from rhodopsin to lipid. Experiments were conducted on bovine rod outer segment (ROS) disks and on recombinant membranes containing lipids with polyunsaturated, docosahexaenoyl (DHA) chains. Poly- and monounsaturated lipids interact specifically with different sites on the rhodopsin surface. Rates of magnetization transfer from protein to DHA are lipid headgroup-dependent and increased in the sequence PC < PS < PE. Boundary lipids are in fast exchange with the lipid matrix on a time scale of milliseconds or shorter. All rhodopsin photointermediates transferred magnetization preferentially to DHA-containing lipids, but highest rates were observed for Meta-III rhodopsin. The experiments show clearly that the surface of rhodopsin has sites for specific interaction with lipids. Current theories of lipid-protein interaction do not account for such surface heterogeneity.