Osmotic stress studies of G-protein-coupled receptor rhodopsin activation.

We summarize and critically review osmotic stress studies of the G-protein-coupled receptor rhodopsin. Although small amounts of structural water are present in these receptors, the effect of bulk water on their function remains uncertain. Studies of the influences of osmotic stress on the GPCR archetype rhodopsin have given insights into the functional role of water in receptor activation. Experimental work has discovered that osmolytes shift the metarhodopsin equilibrium after photoactivation, either to the active or inactive conformations according to their molar mass. At least 80 water molecules are found to enter rhodopsin in the transition to the photoreceptor active state. We infer that this movement of water is both necessary and sufficient for receptor activation. If the water influx is prevented, e.g., by large polymer osmolytes or by dehydration, then the receptor functional transition is back shifted. These findings imply a new paradigm in which rhodopsin becomes solvent swollen in the activation mechanism. Water thus acts as an allosteric modulator of function for rhodopsin-like receptors in lipid membranes.

Sampling Bottleneck in Validating Membrane Dynamics.

Molecular dynamics (MD) simulations have become increasingly impactful in membrane biophysics because they offer atomistic resolution into the atomistic fluctuations of lipid assemblies. Validation of the simulation trajectories with experimental data is crucial for interpretation and application of MD results. As an ideal benchmarking technique, NMR spectroscopy delivers order parameters of the carbon-deuterium bond fluctuations along the lipid chains. Additionally, NMR relaxation can access lipid dynamics providing yet another point for validation of simulation force fields. Here we performed short resampling simulations of membrane trajectories to investigate the lipid CH bond fluctuations on sub-40-ps timescales to explore the local fast dynamics. We recently established a robust framework for analysis of NMR relaxation rates from MD simulations, which improves upon current approaches and shows excellent agreement of experimental and theoretical results. The calculation of relaxation rates from simulations presents a universal challenge that we addressed by hypothesizing the existence of fast CH bond dynamics that evade the analysis of simulation data with temporal resolution of 40 ps (or lower). Indeed, our results support this hypothesis confirming the validity of our solution to the sampling problem. Furthermore, we show that the fast CH bond dynamics occur on timescales at which carbon-carbon bond conformations appear nearly stationary and unaffected by cholesterol. Lastly, we discuss the correspondence to the CH bond dynamics of liquid hydrocarbons and relate their existence to the apparent microviscosity of the bilayer hydrocarbon core.

Biophysics of Membrane Stiffening by Cholesterol and Phosphatidylinositol 4,5-bisphosphate (PIP2).

Cell membranes regulate a wide range of phenomena that are implicated in key cellular functions. Cholesterol, a critical component of eukaryotic cell membranes, is responsible for cellular organization, membrane elasticity, and other critical physicochemical parameters. Besides cholesterol, other lipid components such as phosphatidylinositol 4,5-bisphosphate (PIP2) are found in minor concentrations in cell membranes yet can also play a major regulatory role in various cell functions. In this chapter, we describe how solid-state deuterium nuclear magnetic resonance (2H NMR) spectroscopy together with neutron spin-echo (NSE) spectroscopy can inform synergetic changes to lipid molecular packing due to cholesterol and PIP2 that modulate the bending rigidity of lipid membranes. Fundamental structure-property relations of molecular self-assembly are illuminated and point toward a length and time-scale dependence of cell membrane mechanics, with significant implications for biological activity and membrane lipid-protein interactions.

Molecular simulations and NMR reveal how lipid fluctuations affect membrane mechanics.

Lipid bilayers form the main matrix of functional cell membranes, and their dynamics underlie a host of physical and biological processes. Here we show that elastic membrane properties and collective molecular dynamics (MD) are related by the mean-square amplitudes (order parameters) and relaxation rates (correlation times) of lipid acyl chain motions. We performed all-atom MD simulations of liquid-crystalline bilayers that allow direct comparison with carbon-hydrogen (CH) bond relaxations measured with NMR spectroscopy. Previous computational and theoretical approaches have assumed isotropic relaxation, which yields inaccurate description of lipid chain dynamics and incorrect data interpretation. Instead, the new framework includes a fixed bilayer normal (director axis) and restricted anisotropic motion of the CH bonds in accord with their segmental order parameters, enabling robust validation of lipid force fields. Simulated spectral densities of thermally excited CH bond fluctuations exhibited well-defined spin-lattice (Zeeman) relaxations analogous to those in NMR measurements. Their frequency signature could be fit to a simple power-law function, indicative of nematic-like collective dynamics. Moreover, calculated relaxation rates scaled as the squared order parameters yielding an apparent κC modulus for bilayer bending. Our results show a strong correlation with κC values obtained from solid-state NMR studies of bilayers without and with cholesterol as validated by neutron spin-echo measurements of membrane elasticity. The simulations uncover a critical role of interleaflet coupling in membrane mechanics and thus provide important insights into molecular sites of emerging elastic properties within lipid bilayers.

Cholesterol Stiffening of Lipid Membranes.

Biomembrane order, dynamics, and other essential physicochemical parameters are controlled by cholesterol, a major component of mammalian cell membranes. Although cholesterol is well known to exhibit a condensing effect on fluid lipid membranes, the extent of stiffening that occurs with different degrees of lipid acyl chain unsaturation remains an enigma. In this review, we show that cholesterol locally increases the bending rigidity of both unsaturated and saturated lipid membranes, suggesting there may be a length-scale dependence of the bending modulus. We review our published data that address the origin of the mechanical effects of cholesterol on unsaturated and polyunsaturated lipid membranes and their role in biomembrane functions. Through a combination of solid-state deuterium NMR spectroscopy and neutron spin-echo spectroscopy, we show that changes in molecular packing cause the universal effects of cholesterol on the membrane bending rigidity. Our findings have broad implications for the role of cholesterol in lipid-protein interactions as well as raft-like mixtures, drug delivery applications, and the effects of antimicrobial peptides on lipid membranes.

Phospholipid headgroups govern area per lipid and emergent elastic properties of bilayers.

Phospholipid bilayers are liquid-crystalline materials whose intermolecular interactions at mesoscopic length scales have key roles in the emergence of membrane physical properties. Here we investigated the combined effects of phospholipid polar headgroups and acyl chains on biophysical functions of membranes with solid-state 2H NMR spectroscopy. We compared the structural and dynamic properties of phosphatidylethanolamine and phosphatidylcholine with perdeuterated acyl chains in the solid-ordered (so) and liquid-disordered (ld) phases. Our analysis of spectral lineshapes of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the so (gel) phase indicated an all-trans rotating chain structure for both lipids. Greater segmental order parameters (SCD) were observed in the ld (liquid-crystalline) phase for DPPE-d62 than for DPPC-d62 membranes, while their mixtures had intermediate values irrespective of the deuterated lipid type. Our results suggest the SCD profiles of the acyl chains are governed by methylation of the headgroups and are averaged over the entire system. Variations in the acyl chain molecular dynamics were further investigated by spin-lattice (R1Z) and quadrupolar-order relaxation (R1Q) measurements. The two acyl-perdeuterated lipids showed distinct differences in relaxation behavior as a function of the order parameter. The R1Z rates had a square-law dependence on SCD, implying collective mesoscopic dynamics, with a higher bending rigidity for DPPE-d62 than for DPPC-d62 lipids. Remodeling of lipid average and dynamic properties by methylation of the headgroups thus provides a mechanism to control the actions of peptides and proteins in biomembranes.

Hydration-mediated G-protein-coupled receptor activation.

The Rhodopsin family of G-protein­coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80­100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.

Correction: Flexible lipid nanomaterials studied by NMR spectroscopy.

Correction for 'Flexible lipid nanomaterials studied by NMR spectroscopy' by K. J. Mallikarjunaiah et al., Phys. Chem. Chem. Phys., 2019, 21, 18422-18457, DOI: .

Archerfish respond to a hunting robotic conspecific.

While the unique hunting behavior of archerfish has received considerable scientific attention, the specific social cues that govern behaviors like intraspecific kleptoparasitism in the species are less understood. This paper asks whether the use of a robotic facsimile representing an archerfish can elicit a social response if it approximates an archerfish's appearance, along with key features of its hunting behavior. We found that the fish respond to the robot when it hunted, as indicated by decreasing distances between the robot and fish (and among the fish) during the robot's hunting behavior sequence, as well as higher net transfer entropy when the robot was hunting. These effects were present even when the robot's "hunt" was unproductive and did not result in food. The temporal pattern of fish approach to the robot and each other indicated that the segment of robot hunting behavior proximal to the robotic facsimile shot elicited fish behavior initially. However, earlier cues in the robot's hunting sequence became important following more experience with a food contingency. This indicates that further studies could use a robotic facsimile to conduct a detailed stimulus analysis, changing aspects of the robot's appearance and behavior to uncover the basic mechanisms of information transfer among individuals in a social hunting scenario.

No preference for prosocial helping behavior in rats with concurrent social interaction opportunities.

Helping behavior tasks are proposed to assess prosocial or "empathic" behavior in rodents. This paradigm characterizes the behavior of subject animals presented with the opportunity to release a conspecific from a distressing situation. Previous studies found a preference in rats for releasing restrained or distressed conspecifics over other controls (e.g., empty restrainers or inanimate objects). An empathy account was offered to explain the observed behaviors, claiming subjects were motivated to reduce the distress of others based on a rodent homologue of empathy. An opposing account attributes all previous results to subjects seeking social contact. To dissociate these two accounts for helping behavior, we presented subject rats with three simultaneous choice alternatives: releasing a restrained conspecific, engaging a nonrestrained conspecific, or not socializing. Subjects showed an initial preference for socializing with the nonrestrained conspecific, and no preference for helping. This result contradicts the empathy account, but is consistent with the social-contact account of helping behavior.

Activation of the G-Protein-Coupled Receptor Rhodopsin by Water.

Visual rhodopsin is an important archetype for G-protein-coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent-swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force-based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin-II state. Moreover, we discovered that binding and release of the C-terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration-dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G-protein cycle.

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.

Native Mass Spectrometry Reveals the Simultaneous Binding of Lipids and Zinc to Rhodopsin.

Rhodopsin, a prototypical G-protein-coupled receptor, is responsible for scoptic vision at low-light levels. Although rhodopsin's photoactivation cascade is well understood, it remains unclear how lipid and zinc binding to the receptor are coupled. Using native mass spectrometry, we developed a novel data analysis strategy to deconvolve zinc and lipid bound to the proteoforms of rhodopsin and investigated the allosteric interaction between lipids and zinc binding. We discovered that phosphatidylcholine bound to rhodopsin with a greater affinity than phosphatidylserine or phosphatidylethanolamine, and that binding of all lipids was influenced by zinc but with different effects. In contrast, zinc binding was relatively unperturbed by lipids. Overall, these data reveal that lipid binding can be strongly and differentially influenced by metal ions.

How cholesterol stiffens unsaturated lipid membranes.

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.

Nicotinamide phosphoribosyltransferase purification using SUMO expression system.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via Toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-ß-d-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.

Quantum Mechanical and Molecular Mechanics Modeling of Membrane-Embedded Rhodopsins.

Computational chemistry provides versatile methods for studying the properties and functioning of biological systems at different levels of precision and at different time scales. The aim of this article is to review the computational methodologies that are applicable to rhodopsins as archetypes for photoactive membrane proteins that are of great importance both in nature and in modern technologies. For each class of computational techniques, from methods that use quantum mechanics for simulating rhodopsin photophysics to less-accurate coarse-grained methodologies used for long-scale protein dynamics, we consider possible applications and the main directions for improvement.

Flexible lipid nanomaterials studied by NMR spectroscopy.

Our review addresses how material properties emerge from atomistic-level interactions in the case of lipid membrane nanostructures. We summarize advances in solid-state nuclear magnetic resonance (NMR) spectroscopy in conjunction with alternative small-angle X-ray and neutron scattering methods for investigating lipid flexibility and dynamics. Solid-state 2H NMR is advantageous in that it provides atomistically resolved information about the order parameters and mobility of phospholipids within liquid-crystalline membranes. Bilayer deformation in response to external perturbations occurs over a range of length scales and allows one to disentangle how the bulk material properties emerge from atomistic forces. Examples include structural parameters such as the area per lipid and volumetric thickness together with the moduli for elastic deformation. Membranes under osmotic stress allow one to further distinguish collective undulations and quasielastic contributions from short-range noncollective effects. Our approach reveals how membrane elasticity involves length scales ranging from the bilayer dimensions on down to the size of the flexible lipid segments. Collective lipid interactions of the order of the bilayer thickness and less occur in the liquid-crystalline state. Emergence of lipid material properties is significant for models of lipid-protein forces acting on the mesoscopic length scale that play key roles in biomembrane functions.

Cholesterol Effects on the Physical Properties of Lipid Membranes Viewed by Solid-state NMR Spectroscopy.

In this chapter, we review the physical properties of lipid/cholesterol mixtures involving studies of model membranes using solid-state NMR spectroscopy. The approach allows one to quantify the average membrane structure, fluctuations, and elastic deformation upon cholesterol interaction. Emphasis is placed on understanding the membrane structural deformation and emergent fluctuations at an atomistic level. Lineshape measurements using solid-state NMR spectroscopy give equilibrium structural properties, while relaxation time measurements study the molecular dynamics over a wide timescale range. The equilibrium properties of glycerophospholipids, sphingolipids, and their binary and tertiary mixtures with cholesterol are accessible. Nonideal mixing of cholesterol with other lipids explains the occurrence of liquid-ordered domains. The entropic loss upon addition of cholesterol to sphingolipids is less than for glycerophospholipids, and may drive formation of lipid rafts. The functional dependence of 2H NMR spin-lattice relaxation (R 1Z) rates on segmental order parameters (S CD) for lipid membranes is indicative of emergent viscoelastic properties. Addition of cholesterol shows stiffening of the bilayer relative to the pure lipids and this effect is diminished for lanosterol. Opposite influences of cholesterol and detergents on collective dynamics and elasticity at an atomistic scale can potentially affect lipid raft formation in cellular membranes.