The projects below use analytical ultracentrifugation (AU), light-scattering, and other biophysical analytical tools for the characterization of the viral protein interactions and other protein interacting systems described below. ? ? (1) Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon subunit interactions. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, Gag is in monomer-trimer rather than monomer-dimer equilibrium when IP6 is added. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the """"""""dimer interface"""""""" is essential for the trimeric interaction in IP6. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in 3-dimensional space. Small angle neutron scattering data and molecular modeling provided evidence for a folded structure (see below). A manuscript for the above project was submitted to the Journal and Molecular Biology and received favorable reviews. Suggested revisions are in progress.? ? As indicated above, the gag protein is in monomer-dimer equilibrium in solution. Structural studies from other laboratories have delineated the dimer interface and the amino acid residues that stabilize dimer formation. By mutating two of these residues a mutant Gag protein construct was made that was designated gagWM. Sedimentation equilibrium characterization showed that gagWM remains monomeric at high concentrations in solution. The gag WM mutant construct enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments performed at the NIST Center for Neutron Research of the National Institute of Standards and Technology confirmed its asymmetry and yielded a radius of gyration of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. NIST collaborators constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. A manuscript for the above project was submitted to the Journal and Molecular Biology and received favorable reviews. Suggested revisions are in progress.? ? (2) The Malaria Vacccine Development Branch (MVDB) of NIAID is exploring the use of recombinant Plasmodium falciparum surface proteins for their potential to vaccinate against malaria. We are characterizing selected proteins using AU methodologies.? ? A. One of the major targets of a blood-stage malaria vaccine, the life-cycle stage responsible for clinical disease, is the merozoite surface protein-1 (MSP1). Of particular interest is a primary cleavage product of ~42 kDa (MSP1(42)) derived from the carboxyl end of the MSP1 molecule Several recombinant protein expression systems have been used for production of MSP1(42) and to date, all of the reported recombinant proteins include a 6xHis affinity tag to facilitate purification, including three MSP1(42) clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6xHis tag may not be desirable. The objective of this study was to produce an E. coli derived MSP1(42)-FUP protein without a 6xHis affinity tag at pilot-scale of suitable quality and quantity for preclinical studies and human trials. Biochemical analysis of the final bulk antigen MSP1(42)-FUP protein found that the antigen was more than 99% pure (RP-HPLC) and light scattering, sedimentation velocity and equilibrium characterization found that more than 60% was in the desired monomer state with the balance appearing as predominately a combination of trimers and tretramers. To our knowledge, this is the first recombinant form of MSP1(42) produced without a 6xHis affinity tag suitable for use in humans as a malaria blood-stage vaccine. (Reference 3)? ? B. Merozoite surface protein 3 (MSP3) is a putative antigen for immunization against malaria and this has been demonstrated using a recombinant yeast derived Plasmodium falciparum MSP3 that protected Aotus nancymai monkeys against a virulent challenge infection. In an effort to produce a recombinant MSP3 protein in a scaleable manner, we expressed and purified near full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed highly asymmetrically shaped dimers as determined by analytical size exclusion HPLC with in-line multi-angle light scattering and quasi-elastic light scattering detection and velocity sedimentation) (Rh 7.6 +/- 0.2 nm and 7.3 nm, respectively). Sedimentation equilibrium confirmed the molar mass determined by light scattering. The dimer formation occurred through the leucine zipper-like domain located at the carboxyl-terminus. Evaluation by atomic force microscopy (Albert Jin, DBEPS) identified that the dimer formed star-like molecular structure. A manuscript is in preparation.? ? (3) We have collaborated with the Virology and Vector Core Laboratories of the NIAID Vaccine Research Center on the characterization of Ebola and other viral proteins.? ? The surface glycoprotein (gp) of the Ebola virus is a potential candidate for the development of a vaccine and for structural studies. A recombinant construct of the Ebola gp, expressed in insect cells, was characterized by analytical ultracentrifugation. This construct was found to contain both monomer and trimer forms of the Ebola gp. The trimeric form, which is on the surface of the virus, predominated (~70%). ? ? The bird flu virus has the potential to generate a pandemic infection and this has initiated intense efforts to generate a vaccine. Hemagglutinin (HA) is antigenic glycoprotein found on the surface of all types of influenza viruses. HA is responsible for binding the virus to the cell that is being infected. HA represents an antigen for vaccine development. A set of HA constructs were characterized to evaluate the oligomeric state before they are used in animal studies. The immune response is being correlated with the oligomeric state of the constructs.
