Ayako Egawa

Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR

We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of ≈10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on 13C dipolar spin-diffusion solid-state NMR of uniformly 13C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular CC distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the ≈90 intermolecular distances that BChl c molecules form piggyback-dimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.

Atomic structure of the bacteriochlorophyll c assembly in intact chlorosomes from Chlorobium limicola determined by solid-state NMR

Green sulfur photosynthetic bacteria optimize their antennas, chlorosomes, especially for harvesting weak light by organizing bacteriochlorophyll (BChl) assembly without any support of proteins. As it is difficult to crystallize the organelles, a high-resolution structure of the light-harvesting devices in the chlorosomes has not been clarified. We have determined the structure of BChl c assembly in the intact chlorosomes from Chlorobium limicola on the basis of 13C dipolar spin-diffusion solid-state NMR analysis of uniformly 13C-labeled chlorosomes. About 90 intermolecular C–C distances were obtained by the simultaneous assignment of distance correlations and the structure optimization preceded by the polarization-transfer matrix analysis. An atomic structure was obtained, using these distance constraints. The determined structure of the chlorosomal BChl c assembly is built with the parallel layers of piggyback-dimers. This supramolecular structure would provide insights into the mechanism of weak-light capturing.

Nanodisc-to-Nanofiber Transition of Noncovalent Peptide–Phospholipid Assemblies

We report a novel molecular architecture of peptide–phospholipid coassemblies. The amphiphilic peptide Ac-18A-NH2 (18A), which was designed to mimic apolipoprotein α-helices, has been shown to form nanodisc structures with phospholipid bilayers. We show that an 18A peptide cysteine substitution at residue 11, 18A[A11C], forms fibrous assemblies with 1-palmitoyl-2-oleoyl-phosphatidylcholine at a lipid-to-peptide (L/P) molar ratio of 1, a fiber diameter of 10–20 nm, and a length of more than 1 μm. Furthermore, 18A[A11C] can form nanodiscs with these lipid bilayers at L/P ratios of 4–6. The peptide adopts α-helical structures in both the nanodisc and nanofiber assemblies, although the α-helical bundle structures were evident only in the nanofibers, and the phospholipids of the nanofibers were not lamellar. Fluorescence spectroscopic analysis revealed that the peptide and lipid molecules in the nanofibers exhibited different solvent accessibility and hydrophobicity from those of the nanodiscs. Furthermore, the cysteine substitution at residue 11 did not result in disulfide bond formation, although it was responsible for the nanofiber formation, suggesting that this free sulfhydryl group has an important functional role. Alternatively, the disulfide dimer of 18A[A11C] preferentially formed nanodiscs, even at an L/P ratio of 1. Interconversions of these discoidal and fibrous assemblies were induced by the stepwise addition of free 18A[A11C] or liposomes into the solution. Furthermore, these structural transitions could also be induced by the introduction of oxidative and reductive stresses to the assemblies. Our results demonstrate that heteromolecular lipid–peptide complexes represent a novel approach to the construction of controllable and functional nanoscale assemblies.

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

CHAPTER 3:13C–13C Distance Measurements by Polarization Transfer Matrix Analysis of 13C Spin Diffusion in a Uniformly 13C-Labeled Molecular Complex under Magic Angle Spinning

Spin diffusion of 13C polarization in NMR provides 13C−13C distances under magic angle spinning over a broad spectral width. However, there are difficulties in obtaining the distances accurately in uniformly 13C-labeled molecular complexes in solids. Effects of the weak long-range couplings are suppressed by strong short-range couplings. In addition, direct polarization transfer should be distinguished from relayed transfer. To address these issues, polarization-transfer rate matrix analysis has been applied to the 13C-driven spin diffusion in a uniformly 13C-labeled bacteriochlorophyll c assembly. The transfer rates due to direct dipolar couplings were derived by matrix analysis. Distances were obtained from the rates by perturbation theory for spin diffusion using zero-quantum lineshapes. This procedure gave distances up to 6 A with an accuracy of 25−50%. Correction of the distances from the zero-quantum lineshapes improved the accuracy by about 5−15%. These results show that rate matrix analysis is beneficial for distance analysis of molecular complexes for solid-state NMR. Also, the coefficient and anisotropy of 13C spin diffusion in solids are discussed quantitatively.