Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the ¹⁵N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the ²H solid-state NMR spectra acquired from peptides labelled with 3,3,3-²H₃-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C_α–C_β bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu_1–27 and Influenza A M2_22–46 are shown. Whereas the ¹⁵N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Structure of immune stimulating complex matrices and immune stimulating complexes in suspension determined by small-angle x-ray scattering

Immune stimulating complex (ISCOM) particles consisting of a mixture of Quil-A, cholesterol, and phospholipids were structurally characterized by small-angle x-ray scattering (SAXS). The ISCOM particles are perforated vesicles of very well-defined structures. We developed and implemented a novel (to our knowledge) modeling method based on Monte Carlo simulation integrations to describe the SAXS data. This approach is similar to the traditional modeling of SAXS data, in which a structure is assumed, the scattering intensity is calculated, and structural parameters are optimized by weighted least-squares methods when the model scattering intensity is fitted to the experimental data. SAXS data from plain ISCOM matrix particles in aqueous suspension, as well as those from complete ISCOMs (i.e., with an antigen (tetanus toxoid) incorporated) can be modeled as a polydisperse distribution of perforated bilayer vesicles with icosahedral, football, or tennis ball structures. The dominating structure is the tennis ball structure, with an outer diameter of 40 nm and with 20 holes 5-6 nm in diameter. The lipid bilayer membrane is 4.6 nm thick, with a low-electron-density, 2.0-nm-thick hydrocarbon core. Surprisingly, in the ISCOMs, the tetanus toxoid is located just below the membrane inside the particles.     

Structure and phase behaviour of dimyristoylphosphatidic acid/poly(L-lysine) systems

Summary

Differential scanning calorimetry (DSC) and X-ray diffraction studies on (DMPA)/poly(L-lysine) systems are reported. DSC studies revealed that addition of poly(L-lysine) to DMPA bilayers raises the gel to liquid-crystalline phase transition of the systems, and that this effect depends on the molecular weight of the poly(L-lysine). Small-angle X-ray diffraction measurements showed that, in the liquid-crystalline phase, the lamellar spacing of a DMPA/short-poly(L-lysine) (～4000 mol. wt.) system is shorter than that of a DMPA/long-poly(L-lysine) (～22 000 mol. wt.). In this connection wide-angle X-ray diffraction measurements indicate that the long-poly(L-lysine) adopts a β-sheet conformation on the DMPA bilayers in both the gel and the liquid-crystalline phases, but the short-poly(L-lysine) adopts this conformation only on gel phase DMPA bilayers. We found that the spacings of the hydrocarbon chain packing in a DMPA bilayer in the gel phase increases with temperature, while the spacing between neighbouring polypeptide chains in long-poly(L-lysine) in the β-sheet conformation remains almost constant. These observations indicate that the positively charged lysine residues are structurally independent of the negatively charged head groups of the phospholipid. On the basis of the present results we propose a model to explain the elementary behaviour of extrinsic membrane proteins in biomembranes.     

Characterization of the ground state dynamics of proteorhodopsin by NMR and optical spectroscopies

We characterized the dynamics of proteorhodopsin (PR), solubilized in diC7PC, a detergent micelle, by liquid-state NMR spectroscopy at T?=?323?K. Insights into the dynamics of PR at different time scales could be obtained and dynamic hot spots could be identified at distinct, functionally relevant regions of the protein, including the BC loop, the EF loop, the N-terminal part of helix F and the C-terminal part of helix G. We further characterize the dependence of the photocycle on different detergents (n-Dodecyl ??-D-maltoside DDM; 1,2-diheptanoyl-sn-glycero-3-phosphocholine diC7PC) by ultrafast time-resolved UV/VIS spectroscopy. While the photocycle intermediates of PR in diC7PC and DDM exhibit highly similar spectral characteristics, significant changes in the population of these intermediates are observed. In-situ NMR experiments have been applied to characterize structural changes during the photocycle. Light-induced chemical shift changes detected during the photocycle in diC7PC are very small, in line with the changes in the population of intermediates in the photocycle of proteorhodopsin in diC7PC, where the late O-intermediate populated in DDM is missing and the population is shifted towards an equilibrium of intermediates states (M, N, O) without accumulation of a single populated intermediate.     

Insights on the interactions of synthetic amphipathic peptides with model membranes as revealed by 31P and 2H solid-state NMR and infrared spectroscopies

We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, (31)P and (2)H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. (31)P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas (31)P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, (2)H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length.     

Conformational properties of 2,3-methanopyroglutamic acid in peptides: NMR and x-ray diffraction studies

The effects of replacing L-pyroglutamic acid with the cyclopropane analogue 2,3-methanopyroglutamic acid (2,3-MeGlp) on conformation and enzymatic stability have been investigated in 2,3-MeGlp-NHMe and the novel thyrotropin releasing hormone (TRH) analogue [2,3-MeGlp1]-TRH by x-ray diffraction and nmr. While 2,3-MeGlp-NHMe adopts a folded conformation (small psi angle) in the solid state, several conformations are available to the molecule in solution. 1H-nmr of the diastereomeric mixture [(+/- )-2,3-MeGlp1]-TRH indicates a close orientation of the pyrrolidone and imidazole rings. The 2,3-MeGlp-His amide bond is considerably more stable to pyroglutamate aminopeptidase than the Glp-His bond in TRH.     

NMR and small-angle scattering-based structural analysis of protein complexes in solution

Structural analysis of multi-domain protein complexes is a key challenge in current biology and a prerequisite for understanding the molecular basis of essential cellular processes. The use of solution techniques is important for characterizing the quaternary arrangements and dynamics of domains and subunits of these complexes. In this respect solution NMR is the only technique that allows atomic- or residue-resolution structure determination and investigation of dynamic properties of multi-domain proteins and their complexes. As experimental NMR data for large protein complexes are sparse, it is advantageous to combine these data with additional information from other solution techniques. Here, the utility and computational approaches of combining solution state NMR with small-angle X-ray and Neutron scattering (SAXS/SANS) experiments for structural analysis of large protein complexes is reviewed. Recent progress in experimental and computational approaches of combining NMR and SAS are discussed and illustrated with recent examples from the literature. The complementary aspects of combining NMR and SAS data for studying multi-domain proteins, i.e. where weakly interacting domains are connected by flexible linkers, are illustrated with the structural analysis of the tandem RNA recognition motif (RRM) domains (RRM1-RRM2) of the human splicing factor U2AF65 bound to a nine-uridine (U9) RNA oligonucleotide.     

Structure of the alamethicin pore reconstructed by x-ray diffraction analysis

We reconstructed the electron density profile of the alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the alamethicin pore is of the barrel-stave type consisting of eight alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which alamethicin was tested for its antibacterial activity.     

Acyl chain dynamics of phosphatidylethanolamines containing oleic acid and dihydrosterculic acid: 2H NMR relaxation studies

The dynamical behavior of the acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine has been investigated by using 2H T1 and T2 relaxation times. Lipids were labeled at the 5-,9-,10-, and 16-positions of the sn-2 acyl chain. The profile of deuterium spin-lattice relaxation rate (T1(-1) vs. chain position is characterized in all systems by a marked discontinuity at the positions of the carbon-carbon double bond and the cyclopropane ring; the deuterons at these positions have relaxation rates which are greater than at any other labeled position of the sn-2 chain. For both types of sn-2 acyl chain, assuming a single-exponential correlation time and that the motion is within the rapid regime, the phosphatidylcholine lipid systems are less mobile than their phosphatidylethanolamine analogues. Systems containing an oleoyl chain are more dynamic than their analogues containing a dihydrosterculoyl chain. The rates of motion of the sn-2 acyl chains of phosphatidylethanolamine in a bilayer structure are slower than those of the lipid in an inverted hexagonal structure. In the hexagonal phase, the motional rates of a dihydrosterculoyl chain are slower than those of the corresponding positions of an oleoyl chain.     

Model of interaction between a cardiotoxin and dimyristoylphosphatidic acid bilayers determined by solid-state 31P NMR spectroscopy.

The interaction of cardiotoxin IIa, a small basic protein extracted from Naja mossambica mossambica venom, with dimyristoylphosphatidic acid (DMPA) membranes has been investigated by solid-state 31P nuclear magnetic resonance spectroscopy. Both the spectral lineshapes and transverse relaxation time values have been measured as a function of temperature for different lipid-to-protein molar ratios. The results indicate that the interaction of cardiotoxin with DMPA gives rise to the complete disappearance of the bilayer structure at a lipid-to-protein molar ratio of 5:1. However, a coexistence of the lamellar and isotropic phases is observed at higher lipid contents. In addition, the number of phospholipids interacting with cardiotoxin increases from about 5 at room temperature to approximately 15 at temperatures above the phase transition of the pure lipid. The isotropic structure appears to be a hydrophobic complex similar to an inverted micellar phase that can be extracted by a hydrophobic solvent. At a lipid-to-protein molar ratio of 40:1, the isotropic structure disappears at high temperature to give rise to a second anisotropic phase, which is most likely associated with the incorporation of the hydrophobic complex inside the bilayer.     

Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE-derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root-mean-square deviation of 0.5 A for the well-defined region of the protein, residues 7-37. Many of the side-chains show now well-defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15-21 and 27-29. In solution, the six N-terminal and the two C-terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two-strand beta-sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7-12, adjacent to the beta-sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central beta-sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment-based programs for folding recognition, as opposite to disulfide-bond alignments, reveal new proteins of unrelated sequence and function with a similar structure.     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

The determination of the electron density profile of the human erythrocyte ghost membrane by small-angle x-ray diffraction.

Diffraction patterns of stacked hemolyzed erythrocyte ghosts in the wet state were recorded. Three orders of a surprisingly high first-order periodicity of 600 A were detected. The scattering curves were evaluated by the Q-function method, including lattice distortions of the one-dimensional multilamellar system. The resulting electron density profile of the membrane in the wet state is strongly asymmetric. It consists of an asymmetrical bilayer-type part and an excess of positive relative electron density at the inner, cytoplasmic, side of the membrane. The extension of the whole membrane profile in the wet state is 100--120 A. We suggest that the innermost positive density peak mainly represents the loosely bound protein components spectrin and actin, located at the cytoplasmic side of the membrane and sometimes seen as "fuzzy" material on electron micrographs.     

Molecular organization of human plasma alpha 2-macroglobulin and its trypsin complexes. A small-angle x-ray scattering investigation

The molecular organization of human plasma alpha 2-macroglobulin (alpha 2M), and its 1:1 and 1:2 trypsin complexes, have been investigated using the small-angle x-ray scattering method. All the experimental data can be explained by the same basic model, consisting of three oblate-shaped domains arranged in a sandwich-like structure. Each of the larger peripheral domains consists of two parallel elliptic cylinders associated side-by-side, whereas the smaller central domain consists of just one elliptic cylinder. In the native molecule the three domains are separated by regions of low protein density. Upon trypsin binding the dimensions of the four peripheral cylinders remain unchanged, but their positioning in space is reorganized so that the whole molecule becomes more compact. The model thus offers a plausible explanation for the mechanism of inactivating of the protease by entrapping it between the two larger domains. By comparing the shape and dimensions of the total molecule with those determined for the half-molecular fragment, obtained after reducing the intersubunit disulfide bonds, we propose that the fragment consists of just one of the peripheral domains plus half of the central domain. Different projections of the model are consistent with the electron micrographs of alpha 2M given in the literature. The model can also explain many of the physical and chemical properties recorded for alpha 2M and its protease complexes.     

Tb3+ and Ca2+ binding to phosphatidylcholine. A study comparing data from optical, NMR, and infrared spectroscopies.

The paramagnetic and luminescent lanthanides are unique probes of cation-phospholipid interactions. Their spectroscopic properties provide the means to characterize and monitor complexes formed with lipids in ways not possible with biochemically more interesting cations, such as Ca2+. In this work, Tb3+-phosphatidylcholine complexes are described using the luminescence properties of Tb3+, the effect of its paramagnetism on the 31P NMR and 13C NMR spectra of the lipid, and changes in the infrared spectrum of the lipid induced by the cation. There are two Tb3+-phosphatidylcholine complexes with very different coordination environments, as evidenced by changes in the optical excitation spectrum of the lanthanide. The NMR experiments indicate that the two complexes differ in the number of phosphate groups directly coordinating Tb3+. Tb3+ binding induces changes in the phosphodiester infrared bands that are most consistent with bidentate chelation of Tb3+ by each phosphate, whereas Ca2+-induced changes are more consistent with monodentate coordination. The significance of this discrepancy is discussed.     

Solvated helical backbones: x-ray diffraction study of Boc-Ala-Leu-Aib-Ala-Leu-Aib-OMe.H2O

A second example of insertion of a water molecule into the helical backbone of an apolar peptide is presented here and compared to a similar occurrence in a longer peptide with the same type of sequence of residues, i.e., Boc-Aib-(Ala-Leu-Aib)3-OMe. The backbone of the title compound assumes an approximate 3(10)-helical form with three 4----1 hydrogen bonds. In the place of a fourth 4----1 hydrogen bond, a water molecule is inserted between O(1) and N(4), and acts as a bridge by forming hydrogen bonds N(4) ... W(1) (2.95 A) and W(1) ... O(1) (2.81 A). The water molecule participates in a third hydrogen bond with a neighboring peptide molecule, W(1) ... O(4) (2.91 A). The insertion of the water molecule causes the apolar peptide to mimic an amphiphilic helix. Crystals grown from ethyl acetate/petroleum ether (reported here) or from methanol/water solution are in space group P2(1)2(1)2(1) with a = 12.024(4) A, b = 15.714(6) A, c = 21.411(7) A, Z = 4 and dcalc = 1.124 g/cm3 for C32H58N6O9.H2O. The overall agreement factor R is 6.3% for 2707 reflections observed with intensities greater than 3 sigma(F) and the resolution is 0.90 A.     

Study of the structure of immunoglobins by small-angle x-ray diffraction. I. The structure of IgMCep in solution

The data of small-angle X-ray scattering from monoclonal immunoglobulin MCep (IgM) enabled the shape and geometrical parameters of the molecule in solution at 23 degrees C to be established. The molecule is a flat, strongly anisometric particle with radius of gyration 115 A, volume 1,8 X 10(6) A3, maximum size 380 A, thickness 35-40 A. The most probable molecular model in the approximation of homogeneous electron density in the molecule was suggested, its geometry fitting the experimental parameters. The five IgM subunits are located in the equatorial plane, low-electronic-density regions are located in the centre and at the periphery of the macromolecule. In addition, the absence of fixed angle values between Fab-regions in each subunit is indicative of rather high structural mobility at the periphery of the IgM molecule.     

Molecular dynamics simulations and 2H NMR study of the GalCer/DPPG lipid bilayer

Molecular dynamics simulations were performed on a two-component lipid bilayer system in the liquid crystalline phase at constant pressure and constant temperature. The lipid bilayers were composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules. Two lipid bilayer systems were prepared with GalCer:DPPG ratio 9:1 (10%-DPPG system) and 3:1 (25%-DPPG system). The 10%-DPPG system represents a collapsed state lipid bilayer, with a narrow water space between the bilayers, and the 25%-DPPG system represents an expanded state with a fluid space of approximately 10 nm. The number of lipid molecules used in each simulation was 1024, and the length of the production run simulation was 10 ns. The simulations were validated by comparing the results with experimental data for several important aspects of the bilayer structure and dynamics. Deuterium order parameters obtained from (2)H NMR experiments for DPPG chains are in a very good agreement with those obtained from molecular dynamics simulations. The surface area per GalCer lipid molecule was estimated to be 0.608 +/- 0.011 nm(2). From the simulated electron density profiles, the bilayer thickness defined as the distance between the phosphorus peaks across the bilayer was calculated to be 4.21 nm. Both simulation systems revealed a tendency for cooperative bilayer undulations, as expected in the liquid crystalline phase. The interaction of water with the GalCer and DPPG oxygen atoms results in a strong water ordering in a spherical hydration shell and the formation of hydrogen bonds (H-bonds). Each GalCer lipid molecule makes 8.6 +/- 0.1 H-bonds with the surrounding water, whereas each DPPG lipid molecule makes 8.3 +/- 0.1 H-bonds. The number of water molecules per GalCer or DPPG in the hydration shell was estimated to be 10-11 from an analysis of the radial distribution functions. The formation of the intermolecular hydrogen bonds was observed between hydroxyl groups from the opposing GalCer sugar headgroups, giving an energy of adhesion in the range between -1.0 and -3.4 erg/cm(2). We suggest that this value is the contribution of the hydrogen-bond component to the net adhesion energy between GalCer bilayers in the liquid crystalline phase.