Solid-State NMR Structural and Dynamics Studies of HIV-1 Protein Assemblies
EMSL Project ID
44653
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 850 and 900 MHz solid-state NMR instruments at EMSL, and to a transmission electron microscope. The overall goal of the proposed research is to understand the three-dimensional structure and dynamics of HIV-1 protein assemblies and their interactions with the recently discovered restriction factor protein TRIM5alpha and capsid maturation inhibitors. We focus our attention on CA capsid, CA-SP1, and Gag polyprotein assemblies. Specifically, we will acquire multidimensional homo- and heteronuclear correlation magic angle spinning NMR spectra of HIV-1 protein assemblies of different morphologies and their complexes with TRIM5alpha and capsid maturation inhibitors. The nano-scale architecture will be examined by electron microscopy. Atomic-resolution structure and molecular dynamics on multiple timescales will be derived through the rigorous analysis and numerical simulations of solid-state NMR spectra including application of novel protocols for automated resonance assignments. The goals of this research align directly with the key topical area on understanding dynamics of cellular composition and assembly of multiprotein complexes. The solid-state NMR technology developed through the proposed research will be directly applicable to structural and dynamics analysis of a broad range of multiprotein assemblies at atomic-level resolution.Gag polyprotein from HIV-1 virus is responsible for assembly of virions from infected cells. Gag and its two products, capsid CA protein and capsid-spacer peptide 1 (CA-SP1) are the focus of this research. CA organizes and protects the viral genome by assembling into conical capsids. Following the viral entry into the host, CA disassembles to allow release of the viral genetic material into the host cytoplasmic compartment (processed terms uncoating). CA and the Gag processing intermediate CA-SP1 have recently become attractive targets of HIV-1 uncoating and capsid maturation inhibitors. Despite the promise of targeting CA maturation and uncoating processes by novel inhibitors, the current research is hampered by lack of understanding of the molecular mechanisms of the maturation and uncoating and of their temporal regulation because of the lack of atomic-resolution structural and dynamics information of the assembled Gag, CA, and CA-SP1 interacting with small molecular inhibitors and with TRIM5alpha.
We therefore propose to characterize structurally and dynamically the above HIV-1 protein assemblies using solid-state NMR and transmission electron microscopy. Access to 850 and 900 MHz instruments at EMSL will be critical because of the large size of the protein assemblies under investigation and their high alpha-helical content. This proposal builds on our current proposal entitled "Solid-State NMR Structural and Dynamics Studies of HIV-1 CA Protein Assembly". During the initial very successful proposal period we have prepared the necessary isotopically enriched assemblies of CA, CA-SP1, and Gag, and established the feasibility of high-field multidimensional MAS NMR spectroscopy with excellent-quality spectra acquired for CA assemblies of various morphologies. One manuscript has been published in J. Am. Chem. Soc., and several more are at various stages of preparation.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2011-10-01
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Suiter CL, S Paramasivam, G Hou, S Sun, DM Rice, JC Hoch, DS Rovnyak, and TE Polenova. 2014. "Sensitivity Gains, Linearity, and Spectral Reproducibility in Nonuniformly Sampled Multidimensional MAS NMR Spectra of High Dynamic Range." Journal of Biomolecular NMR 59(2):57-73. doi:10.1007/s10858-014-9824-4