SELF-CROWDING EFFECTS OF THE TWO-DOMAIN RETROVIRAL CAPSID PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). The capsid shell encasing the viral RNA genome is assembled from thousands copies of capsid proteins. However, the HIV-1 capsid protein exists as a dimer in solution, and a double mutation at the interface effectively abolishes the dimerization. Other retroviral capsid proteins characterized to date appear monomeric in solution at low millimolar concentration, and despite the primary sequence divergence the tertiary structures are highly conserved among different species. The capsid protein consists of two domains, namely N- and C- terminal domains. Connected by a five-residue flexible linker, the two domains of capsid protein tumble independently in solution. The primary sequence of the linker is highly conserved among HIV-1 variants, suggesting functional importance and a preferred conformation of the linker. Consistent with the sequence conservation, deuterium exchange using mass spectroscopy delineated a protected region that can be attributed to the interface between HIV-1 capsid N- and C-terminal domains. A recent cryoEM study at nanometer resolution also indicated that the two domains interact with each other in the mature capsid particles. To visualize the transient interactions between the capsid N- and C-terminal domains in solution on a residue-specific level, and to understand how the relative domain motion is affected by protein concentration, we will collect both paramagnetic NMR and small angle X-ray scattering (SAXS) data. We plan to perform SAXS experiments at APS beamline 18-D to assess 1) the concentration dependence of the monomeric Rous sarcoma virus capsid protein, and 2) the concentration dependence of the monomeric mutant of HIV-1capsid protein.