Solid-state 2H NMR Experiments Research Articles

Effect of Melatonin on Lipid Membrane Structure and Membrane Interactions with Amyloid. An NMR and LSPR Study

Alzheimer's Disease (AD) is a neurodegenerative disease and leading cause of dementia resulting in memory loss and cognition decline. The molecular mechanism of AD is not well understood, which has delayed the development of effective treatments. Amyloid-ß (Aß) is the suspected cause of AD, as nonspecific interactions of toxic amyloid oligomers with the plasma membrane cause neuronal cell death. Melatonin is a hormone produced by pineal gland to regulate sleep cycle and has been shown to be protective against Aß in cellular and animal studies, but the molecular mechanism is not well understood. Melatonin can partition into lipid membranes and has remarkable effects on their biophysical properties; this may be important in explaining its protective effects. In this study, we aimed to determine the effect of melatonin on the phase behaviour of model lipid membranes and its ability to inhibit amyloid interaction with lipid bilayers, using nuclear magnetic resonance (NMR) and localized surface plasmon resonance (LSPR), respectively. We used a tertiary mixture of DPPC, POPC, and cholesterol bilayers as a lipid model. Fully hydrated multilamellar lipid stacks were used in solid-state ²H-NMR experiments, showing that melatonin competes with cholesterol to associate with certain lipids and promotes the coexistence of liquid ordered and liquid disordered phases in a temperature-dependent manner. We studied membrane interactions with Aß (1-42) using LSPR and determined that the incorporation of melatonin into the lipid bilayer leads to a decrease in the nonspecific binding of Aß to the supported lipid bilayer. Combining the results from both techniques, we conclude that melatonin's protective effect against amyloid toxicity in lipid membranes is due to its ability to change the structural properties of the membrane and to reduce amyloid affinity to such membranes.

Synthesis, Characterization, and Solid State Dynamic Studies of a Hydrogen Bond-Hindered Steroidal Molecular Rotor with a Flexible Axis.

A novel steroid molecular rotor was obtained in four steps from the naturally occurring spirostane sapogenin diosgenin. The structural and dynamic characterization was carried out by solution NMR, VT X-ray diffraction, solid state 13C CPMAS, and solid state 2H NMR experiments. They allowed the identification of a fast dynamic process with a frequency of 14 MHz at room temperature, featuring a barrier to rotation Ea = 7.87 kcal mol-1. The gathered experimental evidence indicated the presence of a hydrogen bond that becomes stronger as the temperature lowers. This interaction was characterized using theoretical calculations, based on topological analyses of the electronic density and energies. In addition, combining theoretical calculations with experimental measurements, it was possible to propose a partition to Ea (∼8 kcal/mol) into three contributions, that are the cost of the intrinsic rotation (∼2 kcal/mol), the hydrogen bond interaction (∼2 kcal/mol), and the packing effects (∼2-3 kcal/mol). The findings from the present work highlight the relevance of the individual components in the function of molecular machines in the solid state.

Transient Porosity in Densely Packed Crystalline Carbazole-(p-Diethynylphenylene)-Carbazole Rotors: CO2 and Acetone Sorption Properties.

We report for the first time the high sorption properties of a molecular rotor with no permanent voids or channels in its crystal structure. Such crystalline phase originates from THF, DCM, or the irreversible desolvation of entrapped benzene molecules. From these, the benzene in its solvate form acts as rotation stopper, as supported by dynamic characterization using solid-state 2H NMR experiments. In the solvent-free form, the diffusion of small quantities of iodine vapors caused a significant change in the intramolecular rotation, increasing the known activation energy to rotation from 8.5 to 10.6 kcal mol-1. Notably, those results paved the way for the discovery of the high CO2 uptake (201.6 cm3 g-1 at 196 K, under 1 atm) and acetone (5 wt %), a sorption property that was attributed to both, the restriction of the molecular rotation at low temperatures and the flexibility of the molecular axle made of conjugated p-(ethynylphenylene), surrounded by carbazole.

Solid-State 2H NMR Investigation of Transducin Activation by Rhodopsin

X-ray structures of active-state rhodopsin (Meta-II) and the cognate G-protein transducin are available, yet the transducin activation mechanism by rhodopsin is still obscure due to lack of atomistic dynamical information. We are studying the conformations of retinal in active Meta-II, and how the presence of the all-trans retinal agonist yields substantial differences in activation of transducin compared to opsin. Solid-state NMR spectroscopy gives information pertaining both to structures and dynamics, and is a powerful method to study how rhodopsin activates transducin. Experiments are currently underway with selectively deuterated retinal in aligned membrane samples containing active Meta-II [1]. Simulation of the 2H NMR lineshape of the aligned samples in terms of a static uniaxial distribution reveals the bond orientations of retinal methyl groups and mosaic spread, which represents the alignment disorder of the stacked membranes [2,3]. Comparison with the solid-state 2H NMR spectra predicated by published X-ray results enables proposed structures for active Meta-II to be tested [4]. Moreover, the solid-state 2H NMR spectral lineshapes show the role of dynamical fluctuations of the protein. We are also conducting solid-state 2H NMR experiments with deuterated C-terminal peptide of transducin to study the interaction between the G-protein and the rhodopsin transmembrane helices. Our hypothesis is that association and dissociation cycles of transducin depend on the relationship between the local dynamics of peptide and the fluctuations of rhodopsin helices. Solid state 2H NMR experiments can not only tell us how rhodopsin activates transducin, but also can reveal the general mechanisms whereby GPCRs activate the cognate G-proteins. [1] A.V. Struts et al. (2011) PNAS 108, 8263. [2] X. Xu et al. (2014) Encycl. Mag. Res. 3, 275-286. [3] B. Mertz et al. (2012) BBA 1818, 241-251. [4] A.V. Struts et al. (2007) JMB 372, 50-66.

Influence of Glutamic Acid Residues on the Properties of Model Membrane-Spanning Helices

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA- amide) is a constructive model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J.Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged glutamic acid side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments showed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 9.0, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The E16-containing peptide seems to exhibit multi-state behavior at pH 10.9, in bilayers formed by ether-linked lipids, suggesting a pKa that is above pH 9 for the E16 side chain. The rather modest shift in the 2H quadrupolar splittings suggests that the orientation of the transmembrane peptide helix changes rather little at high pH. It is conceivable that the close proximity of E16 to W19 could provide stability to the neutral peptide helix and perhaps influence the pKa of E16. The molecular cousin having E14 instead of E16 shows multi-state behavior from pH 4.0 to pH 10.9 rendering pKa determination enigmatic at this time. Some puzzles remain with respect to comparisons between E16 and E14. We are additionally investigating the peptide-lipid behavior when Glu is introduced in position 12, at the peptide center.

Generalized Model-Free Spectral Density Analysis Applied to Rhodopsin Activation in Membranes

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.

Influence of pH and Side-Chain Negative Charge on the Behavior of Designed Transmembrane Peptides in Lipid Bilayers

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) is a favorable model peptide for investigations of single-residue effects on protein-lipid interactions and the properties of membrane-spanning helices (J. Biol. Chem. 285, 31723). GWALP23 has favorable properties in bilayer membranes because the peptide exhibits only limited dynamic averaging of NMR observables such as the 2H quadrupolar splitting or the 15N-1H dipolar coupling (Biophys. J. 101, 2939). To investigate the potential influence of negatively charged side chains upon system properties, we have substituted a single Leu residue with Glu at different positions and incorporated specific 2H-Ala labels in the core of the single-Trp peptide Y5GWALP23 (see Biochemistry 51, 2044). Solid state 2H NMR experiments were used to examine the peptide orientation and dynamics as functions of the lipid bilayer thickness and pH in hydrated lipid bilayer membranes. We observed well defined 2H quadrupolar splittings for Y5GWALP23-E16 in the pH range from 4.0 to 8.2, suggesting that the peptide helix is well oriented in DOPC lipid bilayers. The glutamic acid residue, though protonated, seemed to confer multi-state behavior at pH 2.5, and the resulting populations exhibited slow exchange on the NMR time scale. The deprotonation of E16 at pH 8.2 did not have any effect on the peptide orientation, perhaps suggesting that the close proximity of E16 to W19 (on the next helical turn) could provide stability to the peptide helix. We are also studying the peptide-lipid behavior when Glu is substituted in position 12 and/or 14, individually or together.

Membrane Interactions of an Acylated and Non-Acylated Lactoferricin Peptide by Solid-State NMR and Fluorescence Spectroscopy and Molecular Dynamics Simulations

LfB6 (RRWQWR-NH2) is a tryptophan- and arginine-rich cationic antimicrobial peptide, derived from bovine lactoferrin, with broad spectrum activity that can be enhanced by N-terminal acylation (CH3(CH2)4CO-RRWQWR-NH2; C6-LfB6). The arginines promote selective interaction with negative charges present in bacterial cell membranes, while the tryptophans prefer the membrane-water interface. We have combined solid-state 2H and 31P NMR and fluorescence experiments with all-atom and coarse-grained molecular dynamics simulations to investigate the interactions of LfB6 and C6-LfB6 peptides in bilayers composed of two distinct compositions: 3:1 POPE:POPG (anionic) and POPC (zwitterionic). Previously we reported that the arginines of C6-LfB6 are first to associate with POPE:POPG; whereas in POPC the C6 tail associates first. Solid-state 2H NMR experimental results confirmed that the lipid order parameters are not significantly changed when C6-LfB6 is bound to negatively-charged membranes, while a slight decrease in order is observed for zwitterionic membranes (Romo, et al. 2011. Biochim. Biophys. Acta. 1808(8):2019-30). We now compare these results with those for non-acylated LfB6 peptide. Solid-state 2H NMR spectra of mechanically aligned samples reveal that the order of POPC and of POPG is reduced in the presence of 1 mol% LfB6. By contrast, POPE shows an increase in order and some line broadening. 31P NMR spectra indicate little effect on the lipid head group for either peptide. The Trp fluorescence emission maxima are blue-shifted for LfB6 (340 nm) and C6-LfB6 (336 nm) in POPE:POPG, compared to POPC (353 nm LfB6 and 348 nm C6-LfB6), suggesting that the Trp residues are less water-exposed in the anionic lipid membranes and when the C6 acyl chain is present.

Experiments Meet Hydrophobic Mismatch: A Re-evaluation Of The Orientation Of Model Transmembrane Peptides From Solid-State NMR

The basic physical rules underlying the organization of biological membranes can be gathered under the simple, but powerful, concept of hydrophobic mismatch. For example, the mutual adjustment of the lipid and protein hydrophobic lengths can be related with the existence of lipid rafts and explain discrete secretory pathways in the Golgi apparatus. The orientation of membrane protein fragments is predicted to follow the same hydrophobic mismatch principles, as illustrated by some experiments and molecular dynamics simulations. However, this appears to be challenged by results of solid-state 2H NMR experiments on model transmembrane peptides, displaying tilt angle values unexpectedly small and weakly reacting to changes of the lipid bilayer thickness.Here we bridge theory and experiments to show that previous 2H NMR experimental data of model transmembrane peptides in membranes of different thickness can be re-interpreted by using alternative models which consider explicit rigid-body peptide fluctuations. The result is a new set of tilts which follows nicely the hydrophobic mismatch expectations, and is coherent with molecular dynamics simulations as well as with other mismatch studies conducted with natural protein fragments.

Is There a Preferential Interaction between Cholesterol and Tryptophan Residues in Membrane Proteins?

Recently, several indications have been found that suggest a preferential interaction between cholesterol and tryptophan residues located near the membrane-water interface. The aim of this study was to investigate by direct methods how tryptophan and cholesterol interact with each other and what the possible consequences are for membrane organization. For this purpose, we used cholesterol-containing model membranes of dimyristoylphosphatidylcholine (DMPC) in which a transmembrane model peptide with flanking tryptophans [acetyl-GWW(LA)8LWWA-amide], called WALP23, was incorporated to mimic interfacial tryptophans of membrane proteins. These model systems were studied with two complementary methods. (1) Steady-state and time-resolved Förster resonance energy transfer (FRET) experiments employing the fluorescent cholesterol analogue dehydroergosterol (DHE) in combination with a competition experiment with cholesterol were used to obtain information about the distribution of cholesterol in the bilayer in the presence of WALP23. The results were consistent with a random distribution of cholesterol which indicates that cholesterol and interfacial tryptophans are not preferentially located next to each other in these bilayer systems. (2) Solid-state 2H NMR experiments employing either deuterated cholesterol or indole ring-deuterated WALP23 peptides were performed to study the orientation and dynamics of both molecules. The results showed that the quadrupolar splittings of labeled cholesterol were not affected by an interaction with tryptophan-flanked peptides and, vice versa, that the quadrupolar splittings of labeled indole rings in WALP23 are not significantly influenced by addition of cholesterol to the bilayer. Therefore, both NMR and fluorescence spectroscopy results independently show that, at least in the model systems studied here, there is no evidence for a preferential interaction between cholesterol and tryptophans located at the bilayer interface.

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment.

Internal motions of d-ribose selectively 2H-labeled at the 2' position were measured using solid state 2H NMR experiments. A sample of d-ribose-2'-d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of -74 and -60 degrees C the C2'-H2' bond was observed to undergo two kinds of motions which were similar to those of C2'-H2'/H2" found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609-8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6+/-0.7 kcal mol(-1), and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy > or =4 kcal mol(-1). At -74 degrees C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at -60 degrees C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2'-H2' bond are most likely from interconversions between C2'-endo and C3'-endo by way of the O4'-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2'-H2' bond within the C2'-endo and C3'-endo potential energy minima.

1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H +-ATPase. Conformational properties and lipid-peptide interactions

PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H +-ATPase, found to regulate the enzyme activity. To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies. High resolution, 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation. Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal. Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions. Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed. Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.

C−C Bond Order Parameters from 2H and 13C Solid-State NMR

In this letter we demonstrate the accuracy of the Ck-1−Ck bond order parameters, , when determined from the Ck−D (2H) bond order parameters, , as experimentally measured on a fully deuterated chain. Two different approaches were taken to determine the C1−C2 bond order parameter, , of a palmitic acid chain (k = 1−16) embedded in a liquid crystal. First, solid-state 2H NMR experiments were performed on the perdeuterated chain in order to obtain its C−D bond order parameter profile. The recursion relation, −2 = + , between consecutive , where k stands for the labeled carbon position was used to calculate . Second, having included a quantity of palmitic acid specifically 13C-labeled at the C1 and C2 positions, the dipolar coupling between these carbons was measured using one- and two-dimensional 13C solid-state NMR spectroscopy. This coupling provides a direct and independent measure of , the order parameter of interest. Both methods agree within 4%.