Solid-State NMR Structural Studies of HIV-1 CA Protein Assembly
EMSL Project ID
30456
Abstract
This is an application in response to the proposal call "Science Theme: Biological Interactions and Dynamics". We request standard, general non-proprietary access to the 900 MHz solid-state NMR instrument at EMSL. Two- and three-dimensional homo- (C-C) and heteronuclear C-N) correlation magic angle spinning NMR spectra of HIV-1 capsid protein CA will be acquired. CA protein organizes and protects the viral genome; the viral entry into the host cell involves disassembly of CA to allow release of the viral genetic material into the host cytoplasmic compartment. The mechanisms of the disassembly and of its temporal regulation are not understood because of lack of atomic-resolution structural information of the assembled capsids. Structural studies of full-length CA by solution NMR and X-ray crystallography have been hampered by its aggregation and difficulties in producing diffraction-quality crystals, despite continuous efforts by multiple investigators. We propose to characterize structurally the HIV-1 CA protein assemblies using solid-state NMR. We will prepare uniformly enriched CA assemblies and perform multidimensional NMR experiments for resonance assignments and structure determination of the CA protein in the assembly. Using differentially enriched CA and protocols developed recently in our laboratory, we will address the structure of the interface of CA assemblies via a combination of multidimensional experiments. Access to 900 MHz instrument at EMSL will be critical because of the large size of CA (25.6 kDa) and of its high helical content. Preliminary 900 MHz C-C DARR spectrum acquired recently at EMSL demonstrate the feasibility of the proposed work and the need for heteronuclear 2- and 3-dimensional experiments. Successful application of solid-state NMR spectroscopy to determine the structure of the full-length HIV-1 CA protein and of the interfaces in the CA assemblies will provide unique insight into the mechanism of capsid formation. Furthermore, in the course of the proposed studies, we envision development of new solid-state NMR methods for characterization of interfaces of capsid assemblies, which will expand the range of protein assemblies amenable to detailed structural analysis.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-08-18
End Date
2011-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Han Y, J Ahn, J Concel, IJL Byeon, AM Gronenborn, J Yang, and TE Polenova. 2010. "Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies." Journal of the American Chemical Society 132(6):1976-1987. doi:10.1021/ja908687k