In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assemblies that are stable and are not affected by magic angle spinning of the samples at frequencies between 10 and 25 kHz. Multidimensional homo- and heteronuclear correlation spectra of CA assemblies of uniformly 13C,15N-labelled CA exhibit narrow lines, indicative of conformational homogeneity of the protein in these assemblies. For the conical assemblies, partial residue-specific resonance assignments were obtained. Analysis of the NMR spectra recorded for the conical and spherical assemblies indicates that the CA protein structure is not significantly different in the different morphologies. The present results demonstrate that the assemblies of CA protein are amenable to detailed structural analysis by solid-state NMR spectroscopy.

Solid-state NMR spectroscopy can be used to probe internal protein dynamics in the absence of the overall molecular tumbling. In this study, we report 15N backbone dynamics in differentially enriched 1-73(U-13C, 15N)/74-108(U-15N) reassembled thioredoxin on multiple timescales using a series of 2D and 3D MAS NMR experiments probing the backbone amide 15N longitudinal relaxation, 1H-15N dipolar order parameters, 15N chemical shift anisotropy (CSA), and signal intensities in the temperature-dependent and 1H T2′ -filtered NCA experiments. The spin-lattice relaxation rates R1(R1 = 1/T1) were observed in the range from 0.012 to 0.64 s-1 indicating large site-to-site variations in dynamics on pico- to nanosecond time scales. The 1H-15N dipolar order parameters, , and 15N CSA anisotropies, δσ reveal the backbone mobilities in reassembled thioredoxin, as reflected in the average = 0.89 ± 0.06 and δσ = 92.3 ± 5.2 ppm, respectively. From the aggregate of experimental data from different dynamics methods, some degree of correlation between the motions on the different time scales has been suggested. Analysis of the dynamics parameters derived from these solid-state NMR experiments indicates higher mobilities for the residues constituting irregular secondary structure elements than for those located in the α-helices and β-sheets, with no apparent systematic differences in dynamics between the α-helical and β-sheet residues. Remarkably, the dipolar order parameters derived from the solid-state NMR measurements and the corresponding solution NMR generalized order parameters display similar qualitative trends as a function of the residue number. The comparison of the solid-state dynamics parameters to the crystallographic B-factors has identified the contribution of static disorder to the B-factors. The combination of longitudinal relaxation, dipolar order parameter, and CSA line shape analyses employed in this study provides snapshots of dynamics and a new insight on the correlation of these motions on multiple time scales.

With the help of CNF conductive bridges, Ti3C2/CNF hybrid particles exhibited significantly enhanced reversible capacity and excellent rate performance.

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Environmental factors such as temperature, hydration, and lipid bilayer properties are tightly coupled to the dynamics of membrane proteins. So far, site-resolved data visualizing the protein's response to alterations in these factors are rare, and conclusions had to be drawn from dynamic data averaged over the whole protein structure. In the current study, high-resolution solid-state NMR at high magnetic field was used to investigate their effects on the molecular dynamics of green proteorhodopsin, a bacterial light-driven proton pump. Through-space and through-bond correlation experiments were employed to identify and characterize highly mobile and motionally restricted regions of proteorhodopsin. Our data show that hydration water plays an essential role for enhancing molecular dynamics of residues in tails and interhelical loops, while it is found less important for residues in transmembrane domains or rigid, structured loop segments. In contrast, switching the lipids from the gel to their liquid crystalline phase enhances molecular fluctuations mainly in transmembrane helices on a time scale of 10(-6) s, but has little effect on loop and tail residues. Increased mobility is especially observed in helices C, F, and G, but also in the EF loop. Fluctuations in those regions are relevant to structural dynamics during the photocycle of proteorhodopsin. Our data are important for the functional understanding of proteorhodopsin, but also offer an important contribution to the general understanding of site-resolved effects of water and lipid bilayers onto the dynamic properties of membrane proteins.

Peptide self-assembly is a hierarchical process, often starting with the formation of α-helices, β-sheets or β-hairpins. However, how the secondary structures undergo further assembly to form higher-order architectures remains largely unexplored. The polar zipper originally proposed by Perutz is formed between neighboring β-strands of poly-glutamine via their side-chain hydrogen bonding and helps to stabilize the sheet. By rational design of short amphiphilic peptides and their self-assembly, here we demonstrate the formation of polar zippers between neighboring β-sheets rather than between β-strands within a sheet, which in turn intermesh the β-sheets into wide and flat ribbons. Such a super-secondary structural template based on well-defined hydrogen bonds could offer an agile route for the construction of distinctive nanostructures and nanomaterials beyond β-sheets.

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Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar−dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances backcalculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure.