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We have demonstrated 57Fe Zero Field Nuclear Magnetic Resonance (ZFNMR) as a powerful tool in determining the structural composition of nickel-cadmium spinel ferrites of various compositions of Ni1-x Cdx Fe2O4 from x = 0 to1, which are synthesized via one-step auto combustion technique. The XRD measurements confirm the phase purity of all the samples. Vibrating Sample Magnetometry (VSM) measurements show that saturation magnetization (MS) increases initially (up to x = 0.3) and then decreases for higher concentrations of cadmium. The Fe3+ ions in the inverted spinel ferrite distribute equally among two possible sites (tetrahedral A and octahedral B) with different hyperfine fields. Therefore for x = 0 under the assumption that Ni enters B sites, 57Fe NMR of Fe3+ ions yield two signals of equal integral intensities in spectral lines corresponding to these sites. Thus, for the sample series Ni1-x Cdx Fe2O4, the contribution of Fe3+ nuclei varies for A and B sub-spectra with the substitution of a non-magnetic Cd2+ ion. By measuring the Fe3+ distribution on A and B sites which is determined from relative spectral areas of A and B NMR sub-spectra the cation distribution is estimated and has been verified by the binomial distribution. Further, XRD Rietveld refinement results are also in good agreement with the composition estimated by NMR technique and the ideal composition. We have demonstrated the usefulness of NMR technique to quantify the accurate composition of the mixed spinel ferrite systems using Ni-Cd ferrite as a test case. Further, the estimated inversion parameter (at around x = 0.4), for the studied system, obtained from ZFNMR, XRD, and VSM techniques are in excellent agreement with each other.

It is well known that cobalt exhibits polymorphism, i.e., the co-existence of both the hcp and fcc phases. In particular, the method of synthesis and other thermodynamic conditions is known to play a crucial role in determining the particular phase of cobalt. In this work, we have compared the phase composition of the cobalt nanoparticles synthesized using two different solvents (water) and ethanol (Co@C). XRD measurements confirm the existence of fcc phase in commercial cobalt nanoparticles (Co@A), co-existence of fcc and hcp phases in Co@B, while the existence of the hcp phase in Co@C. We have studied these cobalt nanoparticles using 59Co internal field nuclear magnetic resonance (IFNMR) for verification of phase composition. Our studies reveal that the Co@A has fcc as a major phase with minor quantity hcp phase. Co@B exhibits approximately equal amount of fcc and hcp phase while Co@C exhibits hcp as a major phase with minor fcc phase. Our SEM micrograph studies confirm that the cobalt particles have spherical shape in the fcc phase. The cobalt particles exhibit both spherical and dendrite morphology confirming the co-existence of fcc and hcp phases, while the sample with pure hcp phase exhibits the dendrite morphology. Our studies also throw light on understanding the effect of solvent in the phase formation of the cobalt nanoparticles.

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.

A borate glasses doped with rare earth Gd3+ ion in the system [6OB2O3 + 30 L12O + x Gd2O3 + (10-x) BiCl3] is prepared by the conventional melt quenching method and their optical properties have been studied. The oxide ion polarizability parameter is calculating by using refractive index of glass materials, which is obtained from UV-Vis spectra. The borate glasses are known to possess high oxide ion polarizability, high refractive index, high basicity and low interaction parameter values. In this present study, theoretical calculation of basicity and interaction parameter, using oxide ion polarization, of the glass network has been addressed. A good linear correlation between the interaction parameter and basicity is observed.

Magnetic nanoparticles of cobalt ferrite are synthesized via a simple reduction route. These synthesized nanoparticles were characterized using x-ray diffraction (XRD), Scanning Electron Microscopy, Raman Spectroscopy, Fourier Transform Infrared studies and their magnetic properties were measured using Vibrating-sample magnetometer. The XRD analysis confirms the formation of single phase CoFe2O4 nanoparticles, with cubic spinel structure, having crystalline size of 5-10 nm, depending on the annealing temperature. Raman spectra analysis confirmed that all the synthesized powders are phase pure. The maximum magnetic saturation of 268 A m(-1) has been observed for the sample calcined at 600 degrees C and correspondingly the magnetic anisotropic constant values are reported. The proposed hydrazine reduction synthesis route is simple in execution and cost effective, which makes it economically adaptable for large scale production of CoFe2O4 nanoparticles.

Correction for ‘Flexible lipid nanomaterials studied by NMR spectroscopy’ by K. J. Mallikarjunaiah et al., Phys. Chem. Chem. Phys., 2019, 21, 18422–18457, DOI: 10.1039/C8CP06179C.

Ferro/ferrimagnetic materials are of fundamental interest due to their variety of applications. The structural and magnetic properties change significantly with different synthesis procedures. Here, we report the synthesis, X-Ray Diffraction (XRD), Vibrating Sample Magnetometry (VSM), and Nuclear Magnetic Resonance (NMR) studies of spinel nickel ferrites doped with non-magnetic cations like cadmium and aluminium. The spinel ferrites like Ni0.7Cd0.3Fe2-xAlxO4 (x = 0, 0.1, 0.2) are synthesized using one step auto combustion technique. The X-ray diffraction measurements confirm the formation of these systems in pure phase. In the present study we have used the modified home-built NMR spectrometer to study 57Fe NMR in these ferro magnetic materials. The difference in the Fe3+ bonding at the octahedral (B-site) and the tetrahedral (A-site) sites result in the different hyperfine fields yielding Internal Field (IF) NMR frequencies at two different frequencies. The substitution of the non-magnetic Al3+ results in increasing in the line width of the NMR spectra corresponding to the octahedral site (B-site). Further, the NMR spectra corresponding to A-site decreases (both in terms of line width and area) due to the decrease in the ferrimagnetic contribution at that site. Change in the local environment around Fe3+ ion present at B-site is very well observed using 57Fe NMR technique.

Oxide glass systems are, interesting because of their bonding like bridging and non -bridging oxygens. Depending on the modifier, the B2O3 glass system can have various Boron -Oxygen network. It is found that, MO modifies the borate network and increases the formation of penta and diborate groups. In this work, we investigated optical properties of lead Borate glass systems (x PbO: (1-x) B2O3) with x varying from 30 - 85 mol % using UV-VIS Spectra and the corresponding band gap was estimated using Tauc relation and these systems behave like direct allowed band gap systems. These results show that, E(g)decreases with the addition of lead content. Further the refractive index measurements also have been carried out at various wavelengths.Many correlation is found between the band gap and refractive index for different compositions. Using different theoretical models a best fit has been tried and Ravindra's relation is found to match with our experimental results.

This article reviews the application of solid-state 2H nuclear magnetic resonance (NMR) spectroscopy for investigating the deformation of lipid bilayers at the atomistic level. For liquid-crystalline membranes, the average structure is manifested by the segmental order parameters (SCD) of the lipids. Solid-state 2H NMR yields observables directly related to the stress field of the lipid bilayer. The extent to which lipid bilayers are deformed by osmotic pressure is integral to how lipid–protein interactions affect membrane functions. Calculations of the average area per lipid and related structural properties are pertinent to bilayer remodeling and molecular dynamics (MD) simulations of membranes. To establish structural quantities, such as area per lipid and volumetric bilayer thickness, a mean-torque analysis of 2H NMR order parameters is applied. Osmotic stress is introduced by adding polymer solutions or by gravimetric dehydration, which are thermodynamically equivalent. Solid-state NMR studies of lipids under osmotic stress probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions. Removal of water yields a reduction of the mean area per lipid, with a corresponding increase in volumetric bilayer thickness, by up to 20% in the liquid-crystalline state. Hydrophobic mismatch can shift protein states involving mechanosensation, transport, and molecular recognition by G-protein-coupled receptors. Measurements of the order parameters versus osmotic pressure yield the elastic area compressibility modulus and the corresponding bilayer thickness at an atomistic level. Solid-state 2H NMR thus reveals how membrane deformation can affect protein conformational changes within the stress field of the lipid bilayer. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (SCD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.

(CH3)4NGeCl3 is prepared, characterized and studied using 1H NMR spin lattice relaxation time and second moment to understand the internal motions and quantum rotational tunneling. Proton second moment is measured at 7 MHz as function of temperature in the range 300-77 K and spin lattice relaxation time (T1) is measured at two Larmor frequencies, as a function of temperature in the range 270-17 K employing a homemade wide-line/pulsed NMR spectrometers. T1 data are analyzed in two temperature regions using relevant theoretical models. The relaxation in the higher temperatures (270-115 K) is attributed to the hindered reorientations of symmetric groups (CH3 and (CH3)4N). Broad asymmetric T1 minima observed below 115 K down to 17 K are attributed to quantum rotational tunneling of the inequivalent methyl groups.

Biomembrane functioning is significantly influenced by the composition and structure of the liquid-crystalline lipid bilayer [1]. Solid-state 2H NMR spectroscopy provides information about atomistic interactions among the membrane constituents by simultaneously probing structure and dynamics [2]. Here we examine the effect of water, osmolytes, cholesterol, and detergents on the liquid-crystalline properties of lipid membranes using NMR relaxation methods. We performed 2H NMR longitudinal (R1Z) and transverse quadrupolar-echo decay (R2QE) experiments on DMPC-d54 bilayers to study membrane lipid dynamics over the time scale ranging from nanoseconds to milliseconds. Plots of R1Z rates or transverse relaxation rates versus squared segmental order parameters (SCD2) show the emergence of collective lipid dynamics [3]. Such a functional behavior characterizes 3-D order-director fluctuations, due to the onset of membrane elasticity [3]. Yet at high hydration, a further R2QE enhancement and confinement of the functional square-law to the segments deeper in the bilayer is seen. Additional contributions from slower dynamics involving water-mediated membrane deformation are evident over mesoscopic length scales on the order of bilayer thickness. Such structural deformations are also evident from bilayer structural parameters calculated using a statistical mean-torque model [4]. The slow dynamics at high hydration or correspondingly low cholesterol or osmolyte concentration are due to modulation of elastic properties of the lipid bilayer. Analysis of the frequency dispersion of the transverse relaxation as a function of such external parameters reveals viscoelastic properties of the liquid-crystalline membranes. Such studies give insights into lipid rafts and membrane composition relevant for biomembrane function. [1] A. Leftin et al. (2014) Biophys. J. 107, 2274[2] K.J. Mallikarjuniah et al. (2011) Biophys. J.100, 98. [3] A. Leftin et al. (2014) eMagRes 4, 199. [4] J.J. Kinnun et al. (2015) BBA 1848, 246.

$(CH_3)_3NHGeCl_3$ is prepared, characterized and studied by $^1H$ NMR and differential scanning calorimetry to understand the molecular dynamics and phase transitions. The proton spin lattice relaxation time $(T_1)$ is measured at 20.53 MHz in the temperature range 391-5.2 K employing a home-made pulsed NMR spectrometer. The $T_1$ results show that $(CH_3)_3NHGeCl_3$ undergoes several phase transitions in the temperature range 391-50 K. Both $T_1$ data and DSC thermograms support the occurrence of phase transitions at 272 K and around 323 K. $T_1$ data are analyzed in three temperature regions using standard models. The $T_1$ variations in the intermediate temperature region 272-100 K are attributed to the hindered reorientations of symmetric groups $(CH_3$ and $(CH_3)_3NH)$. A broad asymmetric $T_1$ minimum in the temperature region 45-20 K is attributed to quantum rotational tunneling of the methyl groups.

Understanding membrane dynamics is crucial to explaining the function of membrane proteins. Phospholipids are commonly employed as model systems to investigate biological membranes. The complex dynamic organization of phospholipid membranes spans several frequency decades, starting from sub-picosecond local motions to millisecond collective dynamics [1]. Such motional frequencies can be accessed using various NMR relaxation methods. To address membrane dynamics mediated by osmotic stress, we measured 2H longitudinal (R1Z) and transverse quadrupolar echo (R2QE) relaxation rates for the liquid-crystalline phase of DMPC-d54 membrane bilayers. Osmotic stress was applied by both dehydration and osmolyte concentration [2]. The R1Z values of individual acyl segments were independent of osmotic stress while the segmental order parameters (SCD) and R1Z profiles followed a theoretical square-law functional dependence [3]. The R2QE rates were found to be sensitive to osmotic pressure as well as the acyl position, thus yielding two important observations: enhanced transverse relaxation rates with increased amount of water per lipid, and limiting lower R2QE values as we dehydrate the membrane. The R2QE rates of the acyl segments and respective SCD values tend to follow a square-law behavior [3] with increasing lipid dehydration. At higher hydration the square-law behavior is limited to those acyl segments deeper in the hydrophobic region, with a break as the head group is approached. These results clearly indicate that water enhances slow cooperative motions whereas they are suppressed by dehydration. Additional complementary Carr-Purcell-Meiboom-Gill (CPMG) dispersion measurements map the frequency dependence of relaxation rates. Such studies in presence of membrane proteins give insight into optimized lipid hydration for their biological functions.[1] A. Leftin et al. (2011) BBA 1808, 818-839.[2] K.J. Mallikarjunaiah et al. (2011) BJ100, 98-107.[3] M.F. Brown (1982) JCP77, 1576-1599.

H-1 NMR spin-lattice relaxation time measurements have been carried out in [(CH3)(4)N](2)SeO4 in the temperature range 389-6.6K to understand the possible phase transitions, internal motions and quantum rotational tunneling. A broad T, minimum observed around 280K is attributed to the simultaneous motions of CH3 and (CH3)(4)N groups. Magnetization recovery is found to be stretched exponential below 72 K with varying stretched exponent. Low-temperature T-1 behavior is interpreted in terms of methyl groups undergoing quantum rotational tunneling. (c) 2007 Elsevier Inc. All rights reserved.

Lipid membrane composition and biophysical properties have substantial influences on cellular functions. Studies of environmental effects on membrane bilayers are a prerequisite for understanding membrane protein functions. Experimental measures of structural parameters like cross-sectional area/lipid of membrane bilayers are vital for molecular dynamics simulations [1,2]. We used solid-state 2H NMR spectroscopy which gives site-specific orientational order parameters of the lipid segments to study structural fluctuations and deformations of phospholipid bilayers due to osmotic stress [1]. Model lipid membrane systems (DMPC and POPC) were subjected to osmotic pressure and temperature to establish their sensitivity to environmental changes in the liquid-crystalline state. Osmotic stress was applied by addition of osmolytes (polyethylene glycol) as well as by gravimetric dehydration. We observed very large changes in segmental order parameters with the application of osmotic pressures in the biological range. The NMR order parameters represent the area/lipid and show large changes in mean-square fluctuations of the lipid structure [3,4]. Stresses from these pressures are thermodynamically equivalent because changing chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to an induced pressure, as verified experimentally [1]. By employing mean-torque analysis of the NMR observables [3] we calculated the mean area per lipid and the volumetric bilayer thickness, which change up to 20% upon introduction of osmotic stress. These 2H NMR studies [4] show striking bilayer deformation due to the application of osmotic pressure. They distinguish molecular-level force regimes associated with lipids that can play a significant role in biological processes.[1] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107. [2] A. Leftin et al. (2011) BBA1808, 818-839. [3] H.I. Petrache et al. (2000) BJ79, 3172-3192. [4] K.J. Mallikarjunaiah et al. (2012) to be submitted to PCCP.

Biological activities of integral membrane proteins depend on the nature of the surrounding lipid bilayer [1]. The dynamic organization of lipid bilayer systems spans a wide frequency range encompassing individual fast acyl motions, molecular rotations, lipid protrusions, and collective bilayer fluctuations [2]. Time scales of these motional modes typically span a large range from sub-picoseconds up to milliseconds, and can be investigated using solid-state NMR spectroscopy. To correlate structural changes of lipid bilayers mediated by osmotic stress [3] with biological function and molecular dynamics, we measured 2H longitudinal (R1Z) and transverse (R2CP) relaxation rates in the liquid-crystalline phase of DMPC-d54 membrane bilayers at various hydration levels. The R1Z experiments used a conventional inversion-recovery pulse sequence, while R2CP rates were measured from quadrupolar-echo intensities using a Carr-Purcell-Meiboom-Gill pulse train. By Fourier transforming individual echoes with different pulse spacings we map the frequency dependence of relaxation rates and motional modes of individual acyl segments. Empirical correlations of acyl chain segmental order parameters (SCD) and R1Zprofiles followed a theoretical square-law functional dependence [4]. Our preliminary results show that R2CP is sensitive to hydration levels as well as the acyl position, thus indicating the presence of slow dynamic modes. The R2CPrates of the acyl segments near the polar head groups increase with lipid dehydration, while the acyl segments deeper in the hydrophobic region show lower R2CP values at high dehydration levels. Similar studies in presence of peptides and proteins can give insight into optimized lipid hydration for protein functionality and slow cooperative motions.[1] M.F. Brown (1994) Chem. Phys. Lipids 73, 159–180.[2] A. Leftin et al. (2011) Biochim. Biophys. Acta 1808, 818–839.[3] K.J. Mallikarjunaiah et al. (2011) Biophys. J. 100, 98–107.[4] M.F. Brown (1982) J. Chem. Phys. 77, 1576–1599.

Intermembrane interactions and forces that govern membrane structure can modulate lipid-protein interactions and thus affect cellular functions [1]. Here we address material properties of the membrane via structural deformation due to external stress using small-angle X-ray scattering (SAXS) and solid-state 2H NMR spectroscopy. These techniques have been extensively used to study structural changes of membrane bilayer dispersions through application of osmotic pressure. However, distinguishing the effects of osmotic stress on intermembrane forces (separation force) versus membrane deformation requires further investigation [2]. We subjected model membranes (DMPC) in the liquid-crystalline state to dehydration and high osmotic pressures (up to 25 MPa). Using SAXS we were able to directly measure the interlamellar spacings and compare the results to solid-state 2H NMR order parameters [1,3]. This approach allowed us to gauge the strength of intermembrane forces for a given hydration state and estimate the area per lipid and structural deformation at the molecular level. Under high osmotic pressure or low hydration we found large area deformations of up to 15% [1]. Also, we verified that the intermembrane force decays exponentially as a function of intermembrane distance as described by the hydration force theory [2]. However, temperature variation revealed decay constants of much larger than a single water molecule, possibly suggesting the existence of forces besides the hydration force. To provide insight into this we have introduced the osmotic coefficient (ratio of work of removing water to thermal energy) which distinguishes regimes of forces. These findings show significant area deformation of membranes and provide insight into the forces that govern intermembrane interactions. [1] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107. [2] V.A. Parsegian et al.. (1979) PNAS76, 2750-2754 [3] H.I. Petrache and M.F. Brown (2007) Meth. Mol. Biol. 400, 341-353.

Cell membrane fluctuations play crucial roles in membrane protein function and cell signaling, yet the specific biophysical mechanisms that control these fluctuations remain poorly understood. Solid-state 2H NMR is a powerful spectroscopic technique that has been used to quantify the free energy penalty of bilayer deformation, as well as the effect of increased osmotic pressure on bilayer properties including the surface area per lipid [1]. These experimental quantities are essential to force-field development and validation for the purpose of accurately modeling lipid bilayers at an atomistic or coarse-grained level. Our hypothesis is that a reformulation of the model used for calculation of segmental order parameters [2] will assist in examination and development of more accurate lipid force fields. This increase in accuracy will in turn lead to more realistic simulations that can provide invaluable insights about membrane bilayer behavior unattainable using other techniques. We carried out a series of concurrent solid-state 2H NMR and molecular dynamics (MD) studies on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, a classical lipid model system, to examine the effect of applied surface tension (from −3 to 5 mJ m−2) on membrane behavior. For both experiment and simulation, as surface tension increased, the surface area per lipid decreased. Segmental order parameters were in general agreement between the two techniques. However, we found that MD simulations using the CHARMM c36 lipid force field tend to underestimate the increase in surface area per lipid with increased surface tension compared to 2H NMR-based results. Interestingly, the MD results match well with previous NMR studies that pre-date the mean-torque model used in our work, demonstrating the interplay between experiment and theory necessary for accurate force field development. [1] K.J. Mallikarjunaiah (2011) BJ 100, 98-107. [2] H.I. Petrache (2000) BJ 79, 3172-3192.

Cellular membrane properties are sensitive to pressure, temperature, and dehydration as well as lipid composition, which can affect function through non-specific lipid-protein interactions [1]. Functional lipid rafts in cellular membranes may correspond to detergent-resistant domains due to the presence of cholesterol. Changes in swelling and stiffening of pure lipid bilayers in the liquid-crystalline phase have been observed [2-4] with addition of detergent and cholesterol. Here we show how structure and associated dynamics of mixed-lipid bilayers are affected by osmotic pressure. Determinations of area per lipid and motional parameters of DMPC membranes in the presence of detergent (C12E8) or cholesterol utilize 2H NMR together with a mean-torque model for interpreting acyl-chain order parameters (SCD) [5]. Swelling by addition of detergent is due to enhanced membrane flexibility, and is counteracted by applying osmotic pressure to the lipid dispersion. By contrast, reduced swelling of multilamellar dispersions due to the stiffening action of cholesterol is reinforced by osmotic pressure. In both cases the membrane area compressibility modulus Ka is calculated from SCD order parameters. We propose that apparent Ka values differ with osmotic pressure for both systems due to changes in the hierarchy of forces and motions. Calculation of the bilayer bending rigidity and the area elastic modulus provides a basis for molecular dynamics simulations of membrane deformations at the atomistic and mesoscopic levels. Osmotic pressure-induced deformation of membranes reveals how lipid-protein interactions can play key roles in biological functions of pressure-sensitive proteins and channels. [1] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107 [2] M.F. Brown et al. (2002) JACS 124, 8471-8484. [3] D. Otten et al. (2000) JPC 104, 12119-12129. [4] G.V. Martinez et al. (2002) PRE 66, 050902. [5] H.I. Petrache et al. (2000) BJ 79, 3172-3192.

Phospholipid membranes are implicated in cellular homeostasis together with a multitude of key biological functions. Many regulatory functions are known to be mediated through protein-lipid interactions. An important feature of pressure-sensitive membrane proteins (mechanosensitive channels, rhodopsin) is that their activation is coupled to membrane tension and curvature elastic stress [1,2]. Solid-state 2H NMR and small-angle X-ray scattering (SAXS) studies of bilayer ensembles of phospholipids under osmotic stress enable membrane structural deformation to be determined. Here we highlight the results from a combined NMR and SAXS approach utilizing pressure-based force techniques that control membrane structure [3] and tension [1].Our 2H NMR results using both osmotic pressure (PEG osmolyte) and gravimetric pressure (low water concentration) techniques show that the segmental order parameters (SCD) of liquid-crystalline DMPC approach very large values ≈0.35 at ≈30 °C. These correspond to ≈20% change in bilayer structural properties (cross-sectional area per lipid and acyl chain thickness) versusthefully hydrated membrane. The two stresses are thermodynamically equivalent because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. A simple theoretical framework based on a unified thermodynamic description is developed. It is shown that the gating threshold for mechanosensitive channels may be shifted to higher or lower values due to lipid-mediated control of channel properties. These findings demonstrate the applicability of solid-state 2H NMR spectroscopy and SAXS together with membrane stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins. [1] S.I. Sukharev et al. (2001) Biophys. J.81, 917-936. [2] A.V. Botelho et al. (2006) Biophys. J.91, 4464-4477. [3] H.I. Petrache, M.F. Brown. (2007) Methods in Membrane Lipids, Humana Press, 339-351.

1H and 19F spin-lattice relaxation times in polycrystalline diammonium hexafluorozirconate have been measured in the temperature range of 10–400 K to elucidate the molecular motion of both cation and anion. Interesting features such as translational diffusion at higher temperatures, molecular reorientational motion of both cation and anion groups at intermediate temperatures and quantum rotational tunneling of the ammonium group at lower temperatures have been observed. Nuclear magnetic resonance (NMR) relaxation time results correlate well with the NMR second moment and conductivity studies reported earlier.

(CH(3))(4)NPF(6) is studied by NMR measurements to understand the internal motions and cross relaxation mechanism between the heterogeneous nuclei. The spin lattice relaxation times (T(1)) are measured for (1)H and (19)F nuclei, at three (11.4, 16.1 and 21.34 MHz) Larmor frequencies in the temperature range 350-50K and (1)H NMR second moment measurements at 7 MHz in the temperature range 300-100K employing home made pulsed and wide-line NMR spectrometers. (1)H NMR results are attributed to the simultaneous reorientations of both methyl and tetramethylammonium groups and motional parameters are evaluated. (19)F NMR results are attributed to cross relaxation between proton and fluorine and motional parameters for the PF(6) group reorientation are evaluated.

Although molecular structures of G-protein-coupled receptors (GPCRs) are becoming increasing available from X-ray crystallography, understanding their functions requires information about molecular dynamics in membranes. Here we use rhodopsin as a model to illuminate general features of GPCR activation. With solid-state 2H NMR spectroscopy we obtain experimental data pertinent to both structure and dynamics. Experimentally, order parameters and relaxation rates are the two observables of solid-state 2H NMR experiments. We propose that the local dynamics of the retinylidene ligand are coupled to large-scale fluctuations of the transmembrane helices of rhodopsin, leading to activation of the receptor. To study the structural dynamics of retinal bound to rhodopsin, we start with an irreducible representation of the correlation function in terms of mean-squared amplitudes and correlation times [1]. The mean-squared amplitudes are related to the orientational order parameter, while the irreducible correlation times include the preexponential factor and energy barrier. To bridge the generalized model-free theory with experimental measurements, we separated the relaxation rates into spectral densities by applying Redfield theory. The spectral densities are Fourier transformation partners of the irreducible correlation functions. By fitting theoretical spectral densities to experimental data we can readily obtain the values of preexponential factors and activation energies [2]. We are currently applying our generalized model-free method to interpret the behavior of active Meta-II rhodopsin. Our aim is to establish if the local fluctuations of the ligand initiate the structural changes of rhodopsin to understand the activation mechanisms of GPCRs in general. Moreover, the results from our generalized model-free analysis method can be used in molecular dynamics (MD) simulations without the limitations of simplified motional models. [1] M.F. Brown (1982) JCP 77, 1576-1599. [2] A.V. Struts et al. (2011) NSMB 18, 392-394.

Crystal structures of rhodopsin are available, yet details of the activation mechanism remain unknown [1,2]. We applied solid-state 2H NMR to investigate structural and dynamical changes occurring in the process of rhodopsin activation. From the 2H NMR spectra, molecular mobility can be obtained by calculating the segmental order parameters from the residual quadrupolar couplings (RQCs). Moreover 2H nuclear spin relaxation rates related to the dynamics can be measured together with the RQCs [2]. Site-specific 2H labels were introduced into different methyl groups of retinal, and relaxation rate measurements were performed as a function of temperature (−30 to −150°C) [3]. Model-free analysis employed an irreducible representation of the combined 2H NMR line shape and relaxation data. Fluctuations of the irreducible components with respect to the average values are characterized by the individual spectral densities of motion evaluated at characteristic frequencies: J0(0), J1(ω0), and J2(2ω0), where ω0 is the nuclear resonance frequency [4]. Differences in the spectral densities manifest details of the methyl group motions within the retinal binding pocket at low temperature. At the high temperature limit, J1(ω0) and J2(2ω0) are insensitive to details of motion and collapse to a universal curve, thus substantiating the validity of the model-free analysis. Further analysis of J1(ω0) and J2(2ω0) involved simultaneous temperature-dependent fitting in terms of both diffusion and jump models for the methyl group dynamics. We conclude that spectral density analysis in terms of fluctuations of nuclear spin Hamiltonian will help us understand the activation mechanism and molecular dynamics of rhodopsin and related G protein-coupled receptors.[1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. [2] M.F. Brown et al. (2010) BBA 1798, 177-193. [3] A.V. Struts et al. (2011) NSMB 18, 392-394. [4] M.F. Brown (1982) JCP 77, 1576-1599.

We address the hypothesis that functions of cellular membranes are affected by non-specific lipid-protein interactions due to bilayer material properties that depend on both pressure and temperature [1]. Changes of either pressure or temperature cause lipid bilayer deformations that are quantified by 2H NMR and X-ray scattering for membranes under osmotic stress. We present measurements of membrane structural parameters such as bilayer thickness and the area per lipid by employing a mean-torque analysis [2-3] of 2H solid-state NMR results together with X-ray scattering data. The 2H NMR experiments for both hydration pressure (low water content) and osmotic pressure (with poly(ethyleneglycol)) show that the segmental order parameters (SCD) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. These two pressures are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure, as experimentally verified by NMR measurements [4]. By considering the equations of state at thermal equilibrium, we extend this approach to address the correspondence between osmotic pressure and hydrostatic pressure. Area per lipid measured using both NMR and X-ray measurements provides a thermodynamic parameter that quantifies membrane deformations [2]. Combined analysis of NMR and X-ray allows us to further test our understanding of dehydration and osmotic stress-induced membrane deformation. We conclude that solid-state 2H NMR spectroscopy and X-ray scattering together with bilayer membrane stress techniques are important tools for understanding of the mechanism of pressure sensitivity of membrane proteins. [1] A.V. Botelho et al. (2006) Biophys. J.91, 4464-4477. [2] H.I. Petrache et al. (2000) Biophys. J.79, 3172-3192. [3] H.I. Petrache et al. (2001) JACS 123, 12611-12622. [4] K.J. Mallikarjunaiah et al. Biophys. J.(in press).

Phospholipid membranes are highly dynamic, ordered structures that involve molecular motions of phospholipids together with collective fluctuations of the bilayer [1]. Membrane structural dynamics on these length scales are sensitive to changes in properties such as temperature, pressure, and chemical potential. Structural deformation accompanying the removal of water from the membrane is well characterized, yet perturbation of membrane dynamics under osmotic stress conditions has not been studied. Here we show that membrane dynamics as revealed by 2H NMR relaxation measurements are sensitive to osmotic stress. Specifically, we measured the segmental order parameters (SCD) and 2H spin-lattice relaxation rates (R1Z) over a broad range of hydration levels. Empirical correlations of acyl chain SCD and R1Z profiles follow a theoretically predicted square-law functional dependence. However, for a given acyl position R1Z is essentially independent of SCD as the hydration water is varied. This is expected if the correlation length of the collective and segmental fluctuations remains unperturbed. The fast segmental fluctuations are decoupled from larger amplitude lipid motions within the osmotically stressed membrane. This result contrasts with studies involving cholesterol, where variations of SCD on the order of those observed in the osmotic stress experiment lead to significant reductions in R1Z rates [2]. In this case, interaction with cholesterol couples local segmental dynamics to collective viscoelastic modes. These results show that the relation of SCD to R1Z is a characteristic marker of lipid matrix composition and collective lipid interactions. Furthermore, our results highlight intrinsic differences in the sensitivity of membrane dynamics, as may be encountered for peripheral protein-membrane interactions and integral membrane-lipid interactions. [1] M.F. Brown and S.I. Chan, Encyclopedia of Nuclear Magnetic Resonance, Wiley, New York 1996, 871-885. [2] G.V. Martinez et al. (2004) Langmuir20,1043-1046.

Cellular functions rely on intermembrane interactions and forces that govern membrane structure and hence modulate lipid-protein interactions [1]. Moreover, the strengths of intermembrane forces vary with interlamellar distances. Here we address material properties of the membrane with structural deformation due to external stress using small-angle X-ray scattering (SAXS) spectroscopy. The SAXS technique has been extensively used to study membrane bilayers through application of osmotic pressure. However, distinguishing the effects of osmotic stress on intermembrane forces (separation force) and membrane deformation requires further investigation [2]. We subjected model membranes (DMPC) in the liquid-crystalline state to dehydration and high osmotic pressures (up to 25 MPa). The work of removal of water from the interlamellar region to the bulk water region restructures the membrane assembly and prompts us to examine membrane properties using complementary techniques. Using SAXS we were able to directly measure the interlamellar spacings and compare the results to solid-state 2H NMR data [1,3]. We correlated the influences of dehydration and osmotic pressure in SAXS results through the interlamellar spacing. This approach allowed us to gauge the strength of intermembrane forces for a given hydration state. The combined techniques allowed us to estimate the area per lipid and structural deformation at the molecular level. Under high osmotic pressure or low hydration we found large area deformations up to 15% [1]. Temperature variation with this approach is used to discern entropic-based forces (lipid protrusions) and ordering-based forces (the hydration force). These findings show significant area deformation of membranes and provide insight into the forces that govern intermembrane interactions.[1] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107.[2] V.A. Parsegian et al. (1979) PNAS 76, 2750-2754.[3] H.I. Petrache and M.F. Brown (2007) Meth. Mol. Biol. 400, 341-353.

Intermembrane forces play a significant role in biological processes such as fusion, shape transformations, and lipid-protein interactions. Forces suggested to govern intermembrane interactions include van der Waals attraction, membrane undulations, hydration force, and lipid protrusions. How do the regimes of these forces overlap and how can we experimentally study them? Through use of osmolytes and dehydration we can control intermembrane spacing in liquid-crystalline DMPC-d54 membranes [1]. Measured order parameters from solid-state 2H NMR spectroscopy allow deformations to be accessed at a molecularly resolved level [2]. Stresses from dehydration and osmotic pressure are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to an induced pressure. A unified theoretical framework predicts an ideal equation of state for the membrane system that depends inversely on the number waters per lipid as confirmed by experimental 2H NMR data [1]. Non-ideal interactions (intermembrane forces) within the membrane system are treated in terms of an osmotic coefficient. Intermembrane forces have differing temperature dependences and can be separated by the temperature variation of the osmotic coefficient. At lower osmotic pressures (larger intermembrane separation) the osmotic coefficient has a linear temperature dependence, agreeing with theoretical predictions for thermal undulations. At high pressures (smaller intermembrane separation) the osmotic coefficient becomes independent of temperature, in accord with predictions for lipid protrusions. Our evidence shows that undulations dominate at intermediate intermembrane distances and protrusions dominate at short distances. We provide a new experimental method for understanding intermembrane forces. This understanding is needed for the interpretation of membrane fusion, shape transformations, and lipid-protein interactions. [1] K.J. Mallikarjunaiah et al. (2011) Biophys. J.100, 98-107. [2] A. Leftin and M.F. Brown (2011) BBA1808, 818-839.