Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy.

The wild-type HIV-1 capsid protein (CA) self-assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV-1. Mass-per-length values of CA assemblies determined by dark-field transmission electron microscopy indicate a variety of structures, ranging from single-wall tubes to multiwall tubes that approximate solid rods. Two-dimensional (2D) solid state (13)C--(13)C and (15)N--(13)C NMR spectra of uniformly (15)N,(13)C-labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3-(13)C(2)-glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N-terminal (NTD) and C-terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self-assembly of full-length CA into tubes. 2D (1)H-(13)C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the alpha-helices of NTD and CTD and from the N- and C-terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.