Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion.

A system based on two designed peptides, namely the cationic peptide K, (KIAALKE)3, and its complementary anionic counterpart called peptide E, (EIAALEK)3, has been used as a minimal model for membrane fusion, inspired by SNARE proteins. Although the fact that docking of separate vesicle populations via the formation of a dimeric E/K coiled-coil complex can be rationalized, the reasons for the peptides promoting fusion of vesicles cannot be fully explained. Therefore it is of significant interest to determine how the peptides aid in overcoming energetic barriers during lipid rearrangements leading to fusion. In this study, investigations of the peptides' interactions with neutral PC/PE/cholesterol membranes by fluorescence spectroscopy show that tryptophan-labeled K∗ binds to the membrane (KK∗ ∼6.2 103 M-1), whereas E∗ remains fully water-solvated. 15N-NMR spectroscopy, depth-dependent fluorescence quenching, CD-spectroscopy experiments, and MD simulations indicate a helix orientation of K∗ parallel to the membrane surface. Solid-state 31P-NMR of oriented lipid membranes was used to study the impact of peptide incorporation on lipid headgroup alignment. The membrane-immersed K∗ is found to locally alter the bilayer curvature, accompanied by a change of headgroup orientation relative to the membrane normal and of the lipid composition in the vicinity of the bound peptide. The NMR results were supported by molecular dynamics simulations, which showed that K reorganizes the membrane composition in its vicinity, induces positive membrane curvature, and enhances the lipid tail protrusion probability. These effects are known to be fusion relevant. The combined results support the hypothesis for a twofold role of K in the mechanism of membrane fusion: 1) to bring opposing membranes into close proximity via coiled-coil formation and 2) to destabilize both membranes thereby promoting fusion.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR.

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

Conformation, self-aggregation, and membrane interaction of peptaibols as studied by pulsed electron double resonance spectroscopy.

Pulsed EPR methods, in particular pulsed electron double resonance (PELDOR) [or double electron-electron resonance (DEER)], are very sensitive to the dipole ··· dipole interaction between electron spins in a pair of free radicals. Using PELDOR, the conformations of a number of double radical-containing biomolecules have been determined. In this review article, we focused our attention on the application of this spectroscopy to nitroxide-labeled peptaibols. This is an emerging class of naturally occurring, relatively short, linear, helical peptide molecules endowed with hydrophobic character, capability to interact with and to alter the structure of membranes, and antibiotic activity. We extracted detailed information on the secondary structures of specifically site-directed, double nitroxide-labeled peptaibols under a variety of experimental conditions, including biologically relevant environments. Moreover, we examined in-depth peptaibol clustering, related to the marked propensity of these molecules to undergo self-association in model and whole-cell membrane systems, using mainly mono-nitroxide-containing synthetic analogs. Finally, based on the PELDOR data accumulated, we proposed models of supramolecular (quaternary) structures of peptaibols and their binding modes to membranes.

Probing coiled-coil assembly by paramagnetic NMR spectroscopy.

Here a new method to determine the oligomeric state and orientation of coiled-coil peptide motifs is described. Peptides K and E, which are designed to form a parallel heterodimeric complex in aqueous solution, were labeled with the aromatic amino acids tryptophan and tyrosine on the C-terminus respectively as 'fingerprint' residues. One of the peptides was also labeled with the paramagnetic probe MTSL. One dimensional proton NMR spectroscopy was used to study the peptide quaternary structure by monitoring the signal suppression of the aromatic labels due to proximity of the nitroxyl radical. 1D-NMR confirmed that the peptides K and E form a heterodimeric coiled coil with a parallel orientation. In addition, fluorescence emission quenching of the aromatic labels due to electron exchange with a nitroxyl radical confirmed the parallel coiled coil orientation. Thus, paramagnetic nitroxide and aromatic fluorophore labeling of peptides yields valuable information regarding the quaternary structure from 1D-NMR and steady-state fluorescence measurements. This convenient method is useful not only to investigate coiled coil assembly, but can also be applied to any defined supramolecular assembly.

Enantiopure isoindolinones through Viedma ripening.

Here we demonstrate that deracemization of isoindolinones using Viedma ripening is possible starting from a racemic mixture of conglomerate crystals. Crystals of the enantiopure isoindolinones lose their chiral identity upon dissolution even without the need for a catalyst. This enabled complete deracemization of the reported isoindolinones without a catalyst.

Spin-echo electron paramagnetic resonance (EPR) spectroscopy of a pore-forming (Lipo)peptaibol in model and bacterial membranes.

This review compiles the unusual structural and dynamic peculiarities of trichogin GA IV and its analogs in lipid bilayers. Different electron spin echo (ESE) spectroscopic techniques were employed to study a set of spin-labeled analogs of trichogin GA IV in model and natural membranes. Pulsed electron-electron double resonance (PELDOR) method enabled the elucidation of the peptide conformation, while the ESE envelope modulation (ESEEM) technique was applied to study the insertion of the site-specifically spin-labeled peptide into the core of the membrane. The latter technique was also used to examine the water accessibility for peptide-attached spin labels at different levels of membrane depth. Finally, it will be shown that measurement of the ESE decays at different temperatures reveals molecular information on the mobility of the transmembrane lipopeptide aggregate. The experimental results are discussed in terms of the antibiotic and toxic activities of trichogin GA IV.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron-electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Structure of self-aggregated alamethicin in ePC membranes detected by pulsed electron-electron double resonance and electron spin echo envelope modulation spectroscopies.

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)(7,18,19)]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70-1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n approximately 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and approximately 5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 approximately TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)(18,19) residues are suggested to stabilize the self-aggregate superstructure.

Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy.

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

Analysis of the amide (15)N chemical shift tensor of the C(alpha) tetrasubstituted constituent of membrane-active peptaibols, the alpha-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues.

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of (15)N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in alpha-aminoisobutyric acid (Aib), the (15)N chemical shift tensor of this C(alpha)-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the (15)N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A (15)N chemical shift-(1)H-(15)N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.

Alamethicin topology in phospholipid membranes by oriented solid-state NMR and EPR spectroscopies: a comparison.

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

PELDOR conformational analysis of bis-labeled alamethicin aggregated in phospholipid vesicles.

Alamethicin (Alm) is a linear peptide antibiotic of great interest for its capability to form self-assembled ion channels in lipid membranes. Here, the pulsed electron-electron double resonance technique was used to obtain unique conformational information on the aggregated peptide in the lipid membrane-bound state. Since a specific helical conformation implies a given length to the peptide molecule, a distance r was measured at the nanometer scale via the electron dipole-dipole interaction between two 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels synthetically incorporated at positions 1 and 16 of this 19-mer peptide. Two data sets were collected (at 77 K): (i) from aggregates of Alm in hydrated egg-yolk phosphocholine (ePC) vesicles (at peptide-to-lipid ratios of 1:200 and 1:75) and (ii) from nonaggregated Alm in pure (nonhydrated) ePC and in solvents of different polarity. The intramolecular distance between the two labels obtained in this manner is in excellent agreement with that calculated on the basis of an almost fully developed alpha-helical conformation for this peptide and is found to be independent of the molecular aggregated state and the environment polarity as well.

Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

The interaction of the complementary K (Ac-(KIAALKE)3-GW-NH2) and E (Ac-(EIAALEK)3-GY-NH2) peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009) is investigated by electron paramagnetic resonance (EPR). By frozen solution continuous-wave EPR and double electron-electron resonance (DEER), the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm) and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.

Enzymatic reaction in a vesicular microreactor: peptaibol-facilitated substrate transport.

Catalytic reactions performed with enzymes localized in lipid vesicles or in whole cells represent a new, promising approach in biocatalysis. The delivery of different substrates into these micro- or nano-'reactors' requires a sufficient permeability of lipid membranes. To increase the permeability of lipid bilayers, one may use different membrane-active peptides, including peptaibols. In the present study, the trypsin-catalyzed hydrolysis of N(alpha)-benzoyl-L-arginine-para-nitroanilide (BAPA; 1) was studied in a phospholipid vesicular system made of phosphatidylcholine (POC), in the presence of the peptaibols alamethicin (ALM) or zervamicin IIB (ZER). Two different manners of compartmentalization of substrate and enzyme (enzyme- vs. substrate-containing vesicles) were used. The kinetics parameters of the reaction in homogeneous solution and in the vesicular systems were determined. The rate of the extra- or intravesicular enzymatic reaction was found to be controlled by substrate diffusion through the lipid bilayer. In comparison with untreated vesicular systems, an up to seven-fold increase in reaction rate was observed in the presence of either ALM or ZER.

Membrane permeabilization of a mammalian neuroendocrine cell type (PC12) by the channel-forming peptides zervamicin, alamethicin, and gramicidin.

Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram-positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1 muM GRAM addition to PC12 cells kept at a membrane potential V(m)=0 mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. -24 mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5-10 microM concentrations while V(m) is kept at -60 mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1-2 min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10-20 min at V(m)=0 mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K(+)-release measurements from a PC12 suspension with a K(+)-selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis-positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell.

Solvent effects on the secondary structure of the membrane-active zervamicin determined by PELDOR spectroscopy.

Zervamicin is a voltage-gated ion-channel-forming peptide. Channels are generally considered to be formed by first insertion of amphipathic molecules into the phospholipid bilayer, followed by self-assembly of a variable number of transmembrane helices. We have studied the length of the peptide structure to address the question whether this peptide is long enough to span the phospholipid bilayer. The pulsed electron-electron double resonance (PELDOR) spectroscopic technique was used to determine the length of the helical molecule in membrane-mimicking solvents. This was achieved from the distance-related dipole-dipole interaction between spin labels, which were located at both ends of the linear peptide chain. The data were obtained by using samples of frozen glassy solutions of MeOH, MeOH/toluene, and MeOH/CHCl(3). Contributions of inter- and intramolecular interactions of spin labels were separated to analyze the intramolecular interaction and the distance distribution function between the labels. It is shown that the main maximum of the distribution functions is located at a distance of ca. 3.3 nm, and this distance appears to be only slightly dependent on the solvent composition. The distribution function was observed to narrow after addition of either CHCl(3) or toluene to MeOH. This effect is rationalized in terms of a decreased mobility of the terminal amino acid residues. By molecular-dynamics simulations, it was shown that the conformation, corresponding with the predominant distance found by PELDOR, agrees well with the mixed alpha/3(10)-helical that was previously determined by NMR. However, in the case toluene was added to the MeOH solution to further increase the hydrophobicity of the environment of the membrane-active peptide, the distribution function gives rise to a minor fraction (7-8%) with a distance of 4.2 nm. This distance corresponds most likely to the more extended 2(7)-helix structure.

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR.

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field.

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.

A 3-D structural model of solid self-assembled chlorophyll a/H(2)O from multispin labeling and MAS NMR 2-D dipolar correlation spectroscopy in high magnetic field.

Magic angle spinning (MAS) NMR with Lee-Goldburg cross-polarization (LG-CP) is used to promote long-range heteronuclear transfer of magnetization and to constrain a structural model for uniformly labeled chlorophyll a/H(2)O. An effective maximum transfer range d(max) can be determined experimentally from the detection of a gradually decreasing series of intramolecular correlations with the (13)C along the molecular skeleton. To probe intermolecular contacts, d(max) can be set to approximately 4.2 A by choosing an LG-CP contact time of 2 ms. Long-range (1)H-(13)C correlations are used in conjunction with carbon and proton aggregation shifts to establish the stacking of the chlorophyll a (Chl a) molecules. First, high-field (14.1 T) 2-D MAS NMR homonuclear ((13)C-(13)C) dipolar correlation spectra provide a complete assignment of the carbon chemical shifts. Second, proton chemical shifts are obtained from (1)H-(13)C heteronuclear dipolar correlation spectroscopy in high magnetic field. The shift constraints and long-range (1)H-(13)C intermolecular correlations reveal a 2-D stacking homologous to the molecular arrangement in crystalline solid ethyl-chlorophyllide a. A doubling of a small subset of the carbon resonances, in the 7-methyl region of the molecule, provides evidence for two marginally different well-defined molecular environments. Evidence is found for the presence of neutral structural water molecules forming a hydrogen-bonded network to stabilize Chl a sheets. In line with the microcrystalline order observed for the rings, the long T(1)'s, and absence of conformational shifts for the (13)C in the phytyl tails, it is proposed that the Chl a form a rigid 3-D space-filling structure. Probably the only way this can be realized with the sheets is by forming bilayers with interpenetration of elongated tails. Such a 3-D space-filling organization of the aggregated Chl a from MAS NMR would match existing models inferred from electron microscopy and low-resolution X-ray powder diffraction, while a micellar model based on neutron diffraction and antiparallel stacking observed in solution can be discarded.