Title

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a hypersensitivity response in N. sylvestris

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Microbiology|Biophysics, General

Degree

Ph.D.

Abstract

The hypersensitivity resistance response directed by the N$\sp\prime$ gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild type coat proteins were compared for variations that may explain N$\sp\prime$ gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and non-elicitor coat proteins. Analytical ultracentrifugation velocity sedimentation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4$\sp\circ$C. In addition, short-column equilibrium sedimentation investigations indicate an increased average molecular weight as a function of coat protein concentration for the R46G coat protein compared to that of the wild type species. Equilibrium sedimentation investigations of the initial stages of self-association show that both coat protein species associate following a monomer-dimer-trimer-hexamer-higher aggregate stoichiometry. The equilibrium sedimentation studies also indicate that the R46G coat protein associates to a higher degree for a given coat protein concentration compared to the wild type species based on the equilibrium constants determined. Viral reconstitution kinetics at 20$\sp\circ$C was used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results show a decreased lag time for reconstitution performed with R46G solutions that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences, indicating that the mechanism of viral assembly is similar for the two coat protein species. Accordingly, an increased rate of formation of 20S component from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20$\sp\circ$C using velocity sedimentation analysis. These conclusions of altered concentrations of coat protein aggregates are consistent with the interpretation that there may be an altered size distribution and/or lifetime of small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus. ^