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1.
J Colloid Interface Sci ; 588: 522-530, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33429348

RESUMO

Nanodiscs are discoidal particles in which a lipid bilayer is encircled by amphipathic molecules such as proteins, peptides, or synthetic polymers. The apolipoprotein-A-I-derived peptide 18A is known to form nanodiscs in the presence of phospholipids, but the detailed mechanism of the formation and deformation of these nanodiscs in response to changes in the surrounding environment is not well understood. Here, we investigated the temperature- and composition-dependent structural changes of 18A-phosphatidylcholine complexes using fluorescence spectroscopy, dynamic light scattering, circular dichroism, static 31P NMR, and electron microscopy. We found that the nanodiscs in fast isotropic rotational motion increased in size above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayers, resulting in the formation of enlarged nanodiscs and a lamellar phase. The lamellar phase was found to be oriented along the magnetic field. Further increase in temperature induced the formation of lipid vesicles. These transformations were explained using a transition model based on the migration of the peptide from the rim of the nanodiscs to the liquid-crystalline bilayer phase. The study outcomes provide a basis for understanding the design principles of discoidal nanostructures for structural biology and nanomedicine applications.


Assuntos
Nanoestruturas , Fosfolipídeos , Bicamadas Lipídicas , Conformação Molecular , Peptídeos , Temperatura
2.
Int J Mol Sci ; 21(11)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32516957

RESUMO

Aqua Gd3+ and Gd-DOTA (gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacete) complexes were studied as a contrast agent in cellular NMR (nuclear magnetic resonance) spectroscopy for distinguishing between intracellular and extracellular spaces. The contrast agents for this purpose should provide strong paramagnetic relaxation enhancement and localize in the extracellular space without disturbing biological functions. Cell membrane permeability to Gd complexes was evaluated from the concentrations of gadolinium complexes in the inside and outside of E. coli cells measured by the 1H-NMR relaxation. The site-specific binding of the complexes to E. coli cells was also analyzed by high-resolution solid-state 13C-NMR. The aqua Gd3+ complex did not enhance T1 relaxation in proportion to the amount of added Gd3+. This Gd3+ concentration dependence and the 13C-NMR indicated that its strong cytotoxicity should be due to the binding of the paramagnetic ions to cellular components especially at the lipid membranes. In contrast, Gd-DOTA stayed in the solution states and enhanced relaxation in proportion to the added amount. This agent exhibited strong T1 contrast between the intra- and extracellular spaces by a factor of ten at high concentrations under which the cells were viable over a long experimental time of days. These properties make Gd-DOTA suitable for selectively contrasting the living cellular space in NMR spectroscopy primarily owing to its weak interaction with cellular components.


Assuntos
Meios de Contraste/química , Complexos de Coordenação/química , Gadolínio/química , Espectroscopia de Ressonância Magnética , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/normas , Escherichia coli/efeitos dos fármacos , Íons/química , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Viabilidade Microbiana/efeitos dos fármacos
3.
Bioorg Med Chem ; 26(21): 5644-5653, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30389410

RESUMO

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.


Assuntos
Proteínas Musculares/metabolismo , Canais de Sódio/metabolismo , Veratridina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Dimiristoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Simulação de Acoplamento Molecular , Proteínas Musculares/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Ratos , Canais de Sódio/química , Veratridina/química
4.
ACS Omega ; 2(6): 2935-2944, 2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31457628

RESUMO

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.

5.
J Biomol NMR ; 64(1): 87-101, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26728076

RESUMO

Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn(2+) mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Cisteína , Metais/química , Modelos Moleculares , Conformação Molecular , Mutação , Proteínas/genética
6.
J Biomol NMR ; 55(2): 189-200, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23271376

RESUMO

Magic-angle-spinning solid-state (13)C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-(13)C,(15)N labeled samples. To overcome this problem, we present a method for assigning (13)C chemical shifts and secondary structures from unresolved two-dimensional (13)C-(13)C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to (13)C(α), (13)C(ß), and (13)C' chemical shifts and cross-peak intensities. The experimental (13)C-(13)C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C(α), C(ß), and C' chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50-200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved (13)C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono , Liofilização , Método de Monte Carlo , Estrutura Secundária de Proteína , Ubiquitina/química
7.
Photosynth Res ; 104(2-3): 221-31, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20063063

RESUMO

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 (13)C dipolar spin-diffusion solid-state NMR analysis of uniformly (13)C-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.


Assuntos
Proteínas de Bactérias/química , Bacterioclorofilas/química , Chlorobium/metabolismo , Ressonância Magnética Nuclear Biomolecular , Organelas/metabolismo , Difusão , Isomerismo , Complexos de Proteínas Captadores de Luz/química
8.
Proc Natl Acad Sci U S A ; 104(3): 790-5, 2007 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-17215361

RESUMO

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 approximately 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 (13)C dipolar spin-diffusion solid-state NMR of uniformly (13)C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular C C distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the approximately 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.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Chlorobium/química , Chlorobium/metabolismo , Ressonância Magnética Nuclear Biomolecular , Chlorobium/efeitos da radiação , Difusão , Dimerização , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
9.
Biochemistry ; 45(24): 7574-85, 2006 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-16768453

RESUMO

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Chlorobium/metabolismo , Proteínas de Bactérias/genética , Bacterioclorofilas/genética , Isótopos de Carbono , Chlorobium/citologia , Chlorobium/genética , Dimerização , Técnicas In Vitro , Isomerismo , Modelos Químicos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular
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