Papers by Trivikram R Molugu
Rhodopsin is a prototype for the large Family A of G-protein–coupled receptors (GPCRs). These pro... more Rhodopsin is a prototype for the large Family A of G-protein–coupled receptors (GPCRs). These proteins regulate many signaling processes, and more than 35% of human pharmaceuticals are targeted against diseases related to dysfunctions of GPCR pathways. Membrane proteins such as GPCRs are challenging to crystallize for X-ray studies. In addition, their effective molar masses in detergent solutions push the limits for solution NMR spectroscopy. By contrast, solid-state NMR allows both the structure and dynamics of membrane proteins to be investigated in a natural lipid bilayer environment. Here, we describe solid-state 2 H NMR methods for investigating structural and dynamical changes of the retinylidene cofactor of the GPCR rhodopsin upon photoillumination. Rhodopsin was regenerated with retinal containing 2 H-labeled C5-, C9-, and C13-methyl groups. The receptor was recombined with phospholipid membranes, which were aligned on planar glass slides. The angular dependences of the 2 H NMR spectra and the corresponding relaxation rates were measured for rhodopsin in the dark and in the cryo-trapped preactive Meta-I and active Meta-II states. Analysis of the 2 H NMR lineshapes using a static uniaxial distribution yields orientational restraints for the retinylidene conformation when bound to the protein. Solid-state 2 H NMR relaxation data provide additional information on the motion of the bound cofactor. The structural and dynamical changes of retinal reveal how its functional groups (methyl groups and the β-ionone ring) affect rhodopsin light
Solid-state deuterium (2H) NMR spectroscopy provides a unique tool for lipid membrane investigati... more Solid-state deuterium (2H) NMR spectroscopy provides a unique tool for lipid membrane investigations. Knowledge of the average structure is obtained from solid-state 2H NMR lineshapes through principal values of the static or motionally averaged coupling tensors due to quadrupolar interactions. For randomly oriented multilamellar lipids or aligned membranes, this technique provides residual quadrupolar couplings (RQC) of the individual C– 2 H labeled segments. The RQC values are used to calculate the segmental order parameters S CD (i) for each segment position (i), which are related to the average membrane
The application of solid-state 2H nuclear magnetic resonance (NMR) spectroscopy gives a powerful ... more The application of solid-state 2H nuclear magnetic resonance (NMR) spectroscopy gives a powerful approach for investigating hydration-mediated effects on lipid bilayer structure and dynamics. The extent to which lipid bilayers are deformed by dehydration stress is inherent to understanding how lipid-protein interactions affect biomembrane functioning. For liquid-crystalline membranes, the average structure is manifested by the segmental order parameters (SCD) of the
lipids. Structural quantities, such as the area per lipid and volumetric bilayer thickness, are obtained by a mean-torque analysis of 2H NMR order parameters. Removal of water in the liquid-crystalline state gives a reduction of the mean area per lipid, together with a corresponding increase in volumetric bilayer thickness. Measurements of order parameters versus osmotic pressure yield the elastic area
compressibility modulus and the corresponding bilayer thickness at an atomistic level. Furthermore, solid-state 2H NMR relaxation rates of lipid bilayers at varying hydration levels afford new insights into the role of water in membrane structural dynamics and viscoelastic properties. Model-free interpretation of spinlattice (R1Z) and transverse (RQE
2 ) relaxation rates suggests that collective chain motions described as order-director fluctuations dominantly contribute to the relaxation. In a continuum picture, elastic deformations in such materials are
collective hydrodynamic phenomena with motional time scales spanning many decades (picoseconds to seconds). The dynamic processes mainly affecting the spin-spin relaxation have characteristic time scales much longer than those contributing to spin-lattice relaxation. Such studies probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions, giving a unique source of information about intermolecular forces pertinent to biomembrane structure and function.
Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this... more Concepts of solid-state NMR spectroscopy and applications to fluid membranes are reviewed in this paper. Membrane lipids with 2 H-labeled acyl chains or polar head groups are studied using 2 H NMR to yield knowledge of their atomistic structures in relation to equilibrium properties. This review demonstrates the principles and applications of solid-state NMR by unifying dipolar and quadrupolar interactions and highlights the unique features offered by solid-state 2 H NMR with experimental illustrations. For randomly oriented multilamellar lipids or aligned membranes, solid-state 2 H NMR enables direct measurement of residual quadrupolar couplings (RQCs) due to individual C− 2 H-labeled segments. The distribution of RQC values gives nearly complete profiles of the segmental order parameters S CD (i) as a function of acyl segment position (i). Alternatively, one can measure residual dipolar couplings (RDCs) for natural abundance lipid samples to obtain segmental S CH order parameters. A theoretical mean-torque model provides acyl-packing profiles representing the cumulative chain extension along the normal to the aqueous interface. Equilibrium structural properties of fluid bilayers and various thermodynamic quantities can then be calculated, which describe the interactions with cholesterol, detergents, peptides, and integral membrane proteins and formation of lipid rafts. One can also obtain direct information for membrane-bound peptides or proteins by measuring RDCs using magic-angle spinning (MAS) in combination with dipolar recoupling methods. Solid-state NMR methods have been extensively applied to characterize model membranes and membrane-bound peptides and proteins, giving unique information on their conformations, orientations, and interactions in the natural liquid-crystalline state. CONTENTS
A B S T R A C T Applications of solid-state NMR spectroscopy for investigating the influences of ... more A B S T R A C T Applications of solid-state NMR spectroscopy for investigating the influences of lipid-cholesterol interactions on membrane fluctuations are reviewed in this paper. Emphasis is placed on understanding the energy landscapes and fluctuations at an emergent atomistic level. Solid-state 2 H NMR spectroscopy directly measures residual quadrupolar couplings (RQCs) due to individual C– 2 H labeled segments of the lipid molecules. Moreover, residual dipolar couplings (RDCs) of 13 C– 1 H bonds are obtained in separated local-field NMR spectroscopy. The distributions of RQC or RDC values give nearly complete profiles of the order parameters as a function of acyl segment position. Measured equilibrium properties of glycerophospholipids and sphingolipids including their binary and tertiary mixtures with cholesterol show unequal mixing associated with liquid-ordered domains. The entropic loss upon addition of cholesterol to sphingolipids is less than for glycerophospholipids and may drive the formation of lipid rafts. In addition relaxation time measurements enable one to study the molecular dynamics over a wide timescale range. For 2 H NMR the experimental spin-lattice (R 1Z) relaxation rates follow a theoretical square-law dependence on segmental order parameters (S CD) due to collective slow dynamics over mesoscopic length scales. The functional dependence for the liquid-crystalline lipid membranes is indicative of viscoelastic properties as they emerge from atomistic-level interactions. A striking decrease in square-law slope upon addition of cholesterol denotes stiffening relative to the pure lipid bilayers that is diminished in the case of lanosterol. Measured equilibrium properties and relaxation rates infer opposite influences of cholesterol and detergents on collective dynamics and elasticity at an atomistic scale that potentially affects lipid raft formation in cellular membranes. ã 2016 Published by Elsevier Ireland Ltd.
Biophysical Journal, 2012
Bone substitutes are increasingly used in orthopedic interventions. Currently, there is a high in... more Bone substitutes are increasingly used in orthopedic interventions. Currently, there is a high interest to optimize the bone scaffolding materials for optimal healing. Using a tibial head defect we investigated bone regeneration using biodegradable poly(lactic-co-glycolic acid) (PLGA) scaffolds, providing a macroporous three-dimensional carrier. Cylindrical scaffolds with similar porosity but different pore sizes of 100-300, 300-500, or 500-710 mm were implanted into a tibial defect of a rat model. Two or four weeks after implantation, the scaffolds were monitored by mMRI and solid-state NMR. In particular, the molecules of the regenerated extracellular matrix (collagen and apatite) were quantitatively studied. Using mMRI, the implanted PLGA scaffolds were clearly visible and a homogeneous generation of ECM was obvious. The regeneration of the collagen moiety was studied by 13 C CPMAS NMR. The total amount of collagen synthesized in the scaffolds depended on the pore size of the scaffolds, best results were obtained for the matrix with 300-500 mm pores. Order parameter measurements of the collagen amino acids showed already very good agreement with those from the natural bone. The inorganic ECM component of the de novo formed bone was investigated by 31 P CPMAS NMR. It could be shown that hydroxyapatite was synthesized in the implant by the chondrocytes. The amount of hydroxyapatite increased significantly towards the end of the 4 week animal study indicative of progressed biomineralization. In all experiments, a pore size of 300 to 500 mm turned out to be most effective. From our molecular assessment, both concentration and molecular dynamics of the de novo formed ECM was already very close to that of native bone. However, as the mMR images revealed, the macroscopic trabecular bone structure in the implants was isotropic as oppose to the anisotropic structure in healthy bone.
Biophysical Journal, 2013
with lipid bilayers are described and understood quite well. Recently, new types of detergents wi... more with lipid bilayers are described and understood quite well. Recently, new types of detergents with cyclohexyl groups or branches in their hydrophobic tails have been synthesized and proposed to be superior for membrane protein studies. Cymal-6 has, for example, been used for isolating membrane proteins such as CCR5 and HIV-1 corepressors. Here we provide a rather comprehensive description of the interactions of Cymal-6 with fluid membranes of POPC. This includes the temperature-dependent phase behavior (i.e., the onset and completion of solubilization), membrane partitioning, disordering, and permeabilization as seen using ITC, time-resolved fluorescence anisotropy of DPH, dynamic light scattering, and the lifetime-based vesicle leakage assay. This study aims to characterize and compare thermodynamic interactions of the lysophospholipid -C12 -lysophosphocholine (lysoPC) and its synthetic analog, n-dodecylphosphocholine (DPC) -with lipid membranes. As a biomolecule possessing detergent-like properties, lysoPC is involved in many biological processes and DPC has been used widely in NMR studies of membrane proteins. We investigate the lipid-detergent systems by determining partition coefficient, mole ratios of bound detergent to lipid at membrane saturation and solubilization boundaries, and the mechanism of membrane disordering and pore formation. Isothermal Titration Calorimetry (ITC) is used for assays such as demicellization, uptake-and-release and solubilization-and-reconstitution. Time-resolved DPH anisotropy and lifetime-based leakage assays are used to study membrane structural changes upon detergent incorporation in liposomes. Both lysoPC and DPC equilibrate with membranes very slowly. We hypothesize that the free energy penalty due to asymmetric membrane insertion limits the membrane uptake of lysoPC and DPC. This would be at variance to other detergents that induce membrane failure above a threshold asymmetry. Results are important for understanding mechanisms for membrane protein isolation and the interactions of amphiphilic biological compounds with lipid membranes.
Biophysical journal, Jan 18, 2014
Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, oft... more Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, ...
Solving high-resolution structures for membrane proteins continues to be a daunting challenge in ... more Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, highresolution NMR experiments on the 13 C, 15 N, 2 H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, H N , CO, C a , and C b chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T 2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.
Spin-lattice relaxation rates (R 1H and R 1F ) of two nuclear species ( 1 H and 19 F) are measure... more Spin-lattice relaxation rates (R 1H and R 1F ) of two nuclear species ( 1 H and 19 F) are measured at different temperatures in the isotropic phase of a liquid crystal (4 -butoxy-3 -fluoro-4isothiocyanatotolane-4OFTOL), over a wide range of Larmor frequency (10 kHz-50 MHz). Their dispersion profiles are found to be qualitatively very different, and the R 1F in particular shows significant dispersion (varying over two orders of magnitude) in the entire isotropic range, unlike R 1H . The proton spin-lattice relaxation, as has been established, is mediated by time modulation of magnetic dipolar interactions with other protons (case of like spins), and the discernable dispersion in the mid-frequency range, observed as the isotropic to nematic transition is approached on cooling, is indicative of the critical slowing of the time fluctuations of the nematic order. Significant dispersion seen in the R 1F extending to very low frequencies suggests a distinctly different relaxation path which is exclusively sensitive to the ultra slow modes apparently present in the system. We find that under the conditions of our experiment at low Zeeman fields, spin-rotation coupling of the fluorine with the molecular angular momentum is the dominant mechanism, and the observed dispersion is thus attributed to the presence of slow torques experienced by the molecules, arising clearly from collective modes. Following the arguments advanced to explain similar slow processes inferred from earlier detailed ESR measurements in liquid crystals, we propose that slowly relaxing local structures representing such dynamic processes could be the likely underlying mechanism providing the necessary slow molecular angular momentum correlations to manifest as the observed low frequency dispersion. We also find that the effects of the onset of cross-relaxation between the two nuclear species when their resonance lines start overlapping at very low Larmor frequencies (below ∼ 400 kHz), provide an additional relaxation contribution.
Nuclear spin-lattice relaxation rate dispersion study of 1 H and 19 F in the isotropic phase of a... more Nuclear spin-lattice relaxation rate dispersion study of 1 H and 19 F in the isotropic phase of a singly fluorinated liquid crystal 4 0 -butoxy-3-fluoro-4-isothiocyantotolane (4OTOLFo) points to their differing relaxation pathways and hence sensitivity to qualitatively different time modulations. In particular fluorine nuclear spins, with strong lattice coupling (larger by two orders) extending to very low frequencies, detect slowly relaxing local structures via the spin-rotation interaction. The field-cycling technique used to carry out these very low frequency measurements, provides for level crossing of the two nuclear species at low enough jump fields, facilitating an additional mechanism of cross-relaxation in the strong coupling limit.
Proton magnetic relaxation dispersion investigations with aqueous solutions of lysozyme and bovin... more Proton magnetic relaxation dispersion investigations with aqueous solutions of lysozyme and bovine serum albumin (BSA) in the 0-5 M range of guanidine hydrochloride (GdnHCl), pH 4.4, 27°C, were taken up with the objective of examining the hydration dynamics of internal cavity waters as the protein is held under increasingly destabilizing conditions. Field cycling NMR and conventional pulsed NMR techniques were employed to cover a frequency range of 100 kHz to 50 MHz. Analyses of dispersion profiles at different concentrations of GdnHCl were carried out considering the contributions from internal and surface waters. The denaturant-dependent variation of internal water contribution indicates that the reorientational disorder of internal waters decreases with increments of the denaturant up to its subdenaturing limit. For both proteins, the variation of effective correlation time with GdnHCl apparently shows a marginal shrink in hydrodynamic volumes under the subdenaturing condition. These results suggest that subdenaturing amounts of GdnHCl restrict the motional freedom of the internal waters, and can have considerable influence on the surface hydration. On increasing the denaturant concentration further, the dispersion amplitude drops sharply, indicating that the chaotropic action of the denaturant now runs over its own cavity water-ordering effect operative in the subdenaturing limit. The results are fundamentally important for the understanding of the susceptibility of protein structure and hydration to denaturants.
Journal of Molecular …, Jan 1, 2011
Rational and in vitro evolutionary approaches to improve either protein stability or aggregation ... more Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named "6B" using multiple rounds of in vitro evolution. T m and optimum activity temperature of 6B is 78°C and 65°C, respectively, which is ∼ 22°C and 30°C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance. .in. † M.Z.K. and S.A. contributed equally to this work.
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Papers by Trivikram R Molugu
lipids. Structural quantities, such as the area per lipid and volumetric bilayer thickness, are obtained by a mean-torque analysis of 2H NMR order parameters. Removal of water in the liquid-crystalline state gives a reduction of the mean area per lipid, together with a corresponding increase in volumetric bilayer thickness. Measurements of order parameters versus osmotic pressure yield the elastic area
compressibility modulus and the corresponding bilayer thickness at an atomistic level. Furthermore, solid-state 2H NMR relaxation rates of lipid bilayers at varying hydration levels afford new insights into the role of water in membrane structural dynamics and viscoelastic properties. Model-free interpretation of spinlattice (R1Z) and transverse (RQE
2 ) relaxation rates suggests that collective chain motions described as order-director fluctuations dominantly contribute to the relaxation. In a continuum picture, elastic deformations in such materials are
collective hydrodynamic phenomena with motional time scales spanning many decades (picoseconds to seconds). The dynamic processes mainly affecting the spin-spin relaxation have characteristic time scales much longer than those contributing to spin-lattice relaxation. Such studies probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions, giving a unique source of information about intermolecular forces pertinent to biomembrane structure and function.
lipids. Structural quantities, such as the area per lipid and volumetric bilayer thickness, are obtained by a mean-torque analysis of 2H NMR order parameters. Removal of water in the liquid-crystalline state gives a reduction of the mean area per lipid, together with a corresponding increase in volumetric bilayer thickness. Measurements of order parameters versus osmotic pressure yield the elastic area
compressibility modulus and the corresponding bilayer thickness at an atomistic level. Furthermore, solid-state 2H NMR relaxation rates of lipid bilayers at varying hydration levels afford new insights into the role of water in membrane structural dynamics and viscoelastic properties. Model-free interpretation of spinlattice (R1Z) and transverse (RQE
2 ) relaxation rates suggests that collective chain motions described as order-director fluctuations dominantly contribute to the relaxation. In a continuum picture, elastic deformations in such materials are
collective hydrodynamic phenomena with motional time scales spanning many decades (picoseconds to seconds). The dynamic processes mainly affecting the spin-spin relaxation have characteristic time scales much longer than those contributing to spin-lattice relaxation. Such studies probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions, giving a unique source of information about intermolecular forces pertinent to biomembrane structure and function.