Abstract
We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.
Highlights
The smallest diameter is roughly equal to that of a truncated icosahedron, or “soccer ball,” that could be formed by 12 R18L-CA pentamers and 20 R18L-CA hexamers
Differences in cross-peak positions among the three two-dimensional 13C-13C correlation spectra in Fig. 3 are minor, despite the large differences in morphology. These results indicate that the underlying molecular conformations of WT-CA and R18L-CA are quite similar in the three samples, and that the number of structurally ordered residues is nearly the same in the three samples
Residues with ⌬rms Ͼ 0.4 ppm are not localized in specific protein segments or at specific regions of intermolecular interactions. These results indicate that conformational differences are relatively minor and distributed over the molecular structures. 15N and 13C chemical shift values were used as input for secondary structure predictions by the TALOSϩ program [49]
Summary
Protein Expression and Purification—WT-CA and R18LCA in pET-11a vector were expressed in Escherichia coli BL21(DE3) cells following the protocol described by Bayro et al [15] Purification was performed according to the published. U-WT-CA and U-R18L-CA are uniformly 13C labeled. 2-Glyc-WT-CA is partially 13C labeled by using [2-13C]glycerol (Glyc) as the carbon source in the protein expression medium. Met-WT-CA is uniformly 15N,13C labeled only at methionine residues. 2-Glyc,Met-R18L-CA is partially 13C labeled with [2-13C]glycerol and simultaneously uniformly 15N,13C labeled at methionine residues Met-WT-CA is uniformly 15N,13C labeled only at methionine residues. 2-Glyc,Met-R18L-CA is partially 13C labeled with [2-13C]glycerol and simultaneously uniformly 15N,13C labeled at methionine residues
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