Evidence for amylose inclusion complexes with multiple acyl chain lipids using solid-state NMR and theoretical approaches.

Amylose is known to form inclusion complexes in the presence of hydrophobic guests. Among lipids, only single-chain fatty acids have been reported as possible guests with the surrounding amylose in a well-defined V-helix conformation. Using experimental 13C solid-state NMR, we studied the formation of inclusion complexes between amylose and a variety of multiple-chains lipids of increasing complexity. Molecular dynamics simulations and calculations of 13C isotropic chemical shifts using the Density Functional Theory approach were performed to support the interpretation of experimental spectra. We provide unambiguous evidences that amylose forms inclusion complexes with lipids bearing multiple acyl chains. Amylose conformations around these lipids are characterized by {ϕ,ψ} anomeric bond dihedral angles near {115°,105°}. In the 13C NMR spectra, this translates into C1 and C4 chemical shifts of 102.5 ppm and 81.1 ppm, regardless of the helical conformation of the amylose surrounding the acyl chains.

Diversity of the Hydrogen Bond Network and Its Impact on NMR Parameters of Amylose B Polymorph: A Study Using Molecular Dynamics and DFT Calculations Within Periodic Boundary Conditions.

Classical molecular dynamics simulations have been combined with quantum (DFT) calculations of 13C NMR parameters in order to relate the experimental spectrum of the double-helix form of the amylose B-polymorph in highly crystalline conditions not only to its 3D structure but also to the arrangement of atoms in the crystal lattice. Structures obtained from these simulations or from geometry optimization procedures at the DFT level have shown the presence of hydrogen bond networks between sugars of the same helix or between residues of the two chains of the double helix. 13C NMR parameter calculations have revealed the impact of such a network on the chemical shifts of carbon atoms. In addition, DFT calculations using periodic boundary conditions were compulsory to highlight the presence of two types of sugar within the crystal sample. It allows us to confirm, theoretically, the experimental hypothesis that the existence of two distinct sugar types in the NMR spectrum is a consequence of crystal packing.

Structures and NMR spectra of short amylose-lipid complexes. Insight using molecular dynamics and DFT quantum chemical calculations.

It is well established that amylose folds in a helix conformation in presence of lipids. Structural features of such molecular complexes are often analysed using 13C NMR spectroscopy. The large size of amylose used to make such analysis doesn't allow to unambiguously correlate structure of polymers and spectroscopic signals. We present structural analysis of small sized amyloses complexed to palmitic acid using classical molecular dynamics. 15 glucoses residues are necessary for the amylose to fold around the palmitic acid in a well-established helix conformation. Simulating 13C NMR spectra using quantum chemical DFT approach, we demonstrate that these spectra are affected by amylose size and specific intramolecular hydrogen bonds. By mean of theoretical NMR spectra of a 19-residues amylose, we precise the attribution of each characteristic resonances. One chemical shift that is usually attributed to a specific carbon may be related to the existence of different inter or intramolecular hydrogen bonds.

Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane.

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

Hydrogen bonding of cholesterol in the lipidic cubic phase.

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.

Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: the four extracellular loops display restricted motion behavior in micelles and in lipid bilayers.

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded ß-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the ß-barrel and loop regions were probed by the spin-lattice relaxation times of the C(α) and C(ß) atoms of the serine and threonine residues, and by cross-polarization dynamics. The ß-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.

Solution-state NMR spectroscopy of membrane proteins in detergent micelles: structure of the Klebsiella pneumoniae outer membrane protein A, KpOmpA.

Structure determination of integral membrane proteins is one of the most important challenges of structural biology. Over the last 7 years, solution-state NMR spectroscopy has become an increasingly useful approach for 3D structure determination and dynamical analysis of membrane proteins solubilized in detergent micelles. We describe herein an ensemble of methods applied in this context, including isotopic labelling, in vitro refolding procedure, and state-of-the-art NMR experiments required for the structure determination of high molecular weight molecular complexes. Furthermore, the basic principles of spectrum interpretation and 3D structure calculation are reported. This approach is illustrated with the structural study of the transmembrane domain of the outer membrane protein A from Klebsiella pneumoniae (KpOmpA).

Modelling the influence of hydrogen bond network on chemical shielding tensors description. GIAO-DFT study of WALP23 transmembrane alpha-helix as a test case.

Density Functional Theory (B3LYP/6-31G(d,p)) calculations of (15)N amide and (13)C carbonyl NMR chemical shielding tensors have been performed on WALP23trans-membrane alpha-helix peptide and compared to solid state NMR experiment performed on [(13)C(1)-Ala(13), (15)N-Leu(14)] specifically labelled peptide powder sample. Using either theoretical results obtained on the whole peptide or experimental data as reference, several simplest chemical models have been explored in order to reduce the computational cost while maintaining good theoretical accuracy. From this study, it appears that the hydrogen bond (N-H...O=C) network that exists in the alpha-helix has a major influence on the chemical shielding tensor and more specifically on the carbonyl (13)C sigma(22) eigenvalue. We show that a small truncated WALP_7 model is not adequate for (13)C(1) NMR description. The application of an external electric field in order to model the hydrogen bond network allows calculating chemical shielding tensors with accurate eigenvalues while the associated eigenvectors are largely modified. Finally, a 23 residues polyglycine peptide that includes the Alanine and Leucine residues for which NMR parameters must be calculated is proposed as the chemical model. This model is sufficient to mostly reproduce the calculation performed on WALP23 with major gain in computational time. Moreover, the application of a low intensity external electric field allows reaching the experimental accuracy for the determination of the eigenvalues.

Order parameters of a transmembrane helix in a fluid bilayer: case study of a WALP peptide.

A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including (13)C and (15)N chemical shift anisotropies and (13)C-(15)N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides ((2)H, (13)C, and (15)N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 degrees was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 degrees previously determined from (2)H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error.

Structural properties of a peptide derived from H+ -V-ATPase subunit a.

The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H(+)-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously suggested based on NMR studies of a similar peptide in DMSO. The pKa of both histidine residues that are important for proton transport was measured in water and in SDS. The differences that are found demonstrate that the histidine residues interact with the SDS polar heads. In detergent, circular dichroism data indicate that the secondary structure of the peptide depends on the pH and the type of detergent used. Using solid-state NMR, it is shown that the peptide is immobile in phospholipid bilayers, which means that it is probably not a single transmembrane helix in these samples. The environment is important for the structure of TM7, so in subunit a it is probably held in place by the other transmembrane helices of this subunit.

Solution state NMR structure and dynamics of KpOmpA, a 210 residue transmembrane domain possessing a high potential for immunological applications.

The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR.This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins(210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly 15N,13C,2H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 A for the main chain atoms in the core eight-stranded beta-barrel. The protein dynamics was assessed, in a residue-specific manner, by 1H-15N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and 1H-2H chemical exchange (hour-weeks).

Acyl chain order parameter profiles in phospholipid bilayers: computation from molecular dynamics simulations and comparison with 2H NMR experiments.

Order parameters from deuterium NMR are often used to validate or calibrate molecular dynamics simulations. This paper gives a short overview of the literature in which experimental order parameters from (2)H NMR are compared to those calculated from MD simulations. The different ways in which order parameters from experiment are used to calibrate and validate simulations are reviewed. In the second part of this review, a case study of cholesterol in a DMPC bilayer is presented. It is concluded that the agreement between experimental data and simulation is favorable in the hydrophobic region of the membrane, for both the phospholipids and cholesterol. In the interfacial region the agreement is less satisfactory, probably because of the high polarity of this region which makes the correct computation of the electrostatics more complex.

Temperature and timescale dependence of protein dynamics in methanol : water mixtures.

Experimental and computer simulation studies have suggested the presence of a transition in the dynamics of hydrated proteins at around 180-220 K. This transition is manifested by nonlinear behaviour in the temperature dependence of the average atomic mean-square displacement which increases at high temperature. Here, we present results of a dynamic neutron scattering analysis of the transition for a simple enzyme: xylanase in water : methanol solutions of varying methanol concentrations. In order to investigate motions on different timescales, two different instruments were used: one sensitive to approximately 100 ps timescale motions and the other to approximately ns timescale motions. The results reveal distinctly different behaviour on the two timescales examined. On the shorter timescale the dynamics are dictated by the properties of the surrounding solvent: the temperature of the dynamical transition lowers with increasing methanol concentration closely following the melting behaviour of the corresponding water : methanol solution. This contrasts with the longer (ns) timescale results in which the dynamical transition appears at temperatures lower than the corresponding melting point of the cryosolvent. These results are suggested to arise from a collaborative effect between the protein surface and the solvent which lowers the effective melting temperature of the protein hydration layer. Taken together, the results suggest that the protein solvation shell may play a major role in the temperature dependence of protein solution dynamics.

Understanding sterol-membrane interactions part I: Hartree-Fock versus DFT calculations of 13C and 1H NMR isotropic chemical shifts of sterols in solution and analysis of hydrogen-bonding effects.

1H and 13C NMR chemical shifts are exquisitely sensitive probes of the local environment of the corresponding nuclei. Ultimately, direct determination of the chemical shifts of sterols in their membrane environment has the potential to reveal their molecular interactions and dynamics, in particular concerning the hydrogen-bonding partners of their OH groups. However, this strategy requires an accurate and efficient means to quantify the influence of the various interactions on chemical shielding. Herein the validity of Hartree-Fock and DFT calculations of the 13C and 1H NMR chemical shifts of cholesterol and ergosterol are compared with one another and with experimental chemical shifts measured in solution at 500 MHz. A computational strategy (definition of basis set, simpler molecular models for the sterols themselves and their molecular complexes) is proposed and compared with experimental data in solution. It is shown in particular that the effects of hydrogen bonding with various functional groups (water as a hydrogen-bond donor and acceptor, acetone) on NMR chemical shifts in CDCl3 solution can be accurately reproduced with this computational approach.

Understanding sterol-membrane interactions, part II: complete 1H and 13C assignments by solid-state NMR spectroscopy and determination of the hydrogen-bonding partners of cholesterol in a lipid bilayer.

The complete assignment of cholesterol 1H and 13C NMR resonances in a lipid bilayer environment (Lalpha-dimyristoylphosphatidylcholine/cholesterol 2:1) has been obtained by a combination of 1D and 2D MAS NMR experiments: 13C spectral editing, ge-HSQC, dipolar HETCOR and J-based HETCOR. Specific chemical shift variations have been observed for the C1-C6 atoms of cholesterol measured in CCl4 solution and in the membrane. Based on previous work (F. Jolibois, O. Soubias, V. Reat, A. Milon, Chem. Eur. J. 2004, 10, preceding paper in this issue: DOI: 10.1002/chem.200400245) these variations were attributed to local changes around the cholesterol hydroxy group, such as the three major rotameric states of the C3-O3 bond and different hydrogen bonding partners (water molecules, carboxy and phosphodiester groups of phosphatidylcholine). Comparison of the experimental and theoretical chemical shifts obtained from quantum-chemistry calculations of various transient molecular complexes has allowed the distributions of hydrogen bonding partners and hydroxy rotameric states to be determined. This is the first time that the probability of hydrogen bonding occurring between cholesterol's hydroxy group and phosphatidylcholine's phosphodiester has been determined experimentally.

15N T2' relaxation times of bacteriorhodopsin transmembrane amide nitrogens.

15N T(2)' relaxation times of bacteriorhodopsin (BR) amide nitrogens were determined in the temperature range from 40 to -60 degrees C using a Hahn echo pulse sequence and proton decoupling during the echo and detection times. Using oriented membrane samples, with their bilayer normal parallel to the external magnetic field, the (15)N amide nitrogens belonging to the transmembrane helices could be selected for the analysis. The experiments were performed on purple membrane fragments (in which BR is organized in a 2D crystalline network) and on BR reconstituted into dimyristoylphosphatidylcholine at a 1:150 molar ratio (in which BR is in a freely diffusing monomeric state at 40 degrees C and in an aggregated state at 4 degrees C and below). The results are discussed in terms of helix dynamics, mosaic spread and resolution of the (15)N spectra for the various samples and experimental conditions.

High resolution 2D 1H-13C correlation of cholesterol in model membrane.

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.