During the morphogenesis of double-stranded DNA bacteriophages, a DNA-free protein capsid (procapsid) is assembled and subsequently packages the bacteriophage DNA. Procapsids consist of a multimolecular outer shell and two classes of internal proteins that assist, by unknown mechanisms, assembly of the outer shell. Previous studies of the structure and assembly of viruses suggest that assembly of outer procapsid shells kinetically controlled by induction of changes in the conformation in unassembled subunits. By this hypothesis, unassembled subunits in a nonassociating conformation switch to a conformation appropriate for entry into the assembly pathway after contact with a partially assembled precursor of the procapsid (conformational switching). However, details of the extent to which conformational switching occurs are not known for any viral capsid. The long-range goal of the research proposed here is to determine the mechanisms by which procapsids assemble from unassembled proteins. Emphasis is to be placed on determining how internal proteins assist assembly of the procapsid's outer shell and on determining the extent to which assembly is controlled by conformational switching. To reach this goal, the specific aims are to accomplish the following, using the related bacteriophages, T7 and T3, as models: (a) increase understanding of the structure of procapsids; electron microscopy, immuno-electron microscopy, protein-protein crosslinking, controlled proteolysis (based on our previous determination of tertiary structure by this procedure) and laser-Raman spectroscopy will be used, (b) detect and isolate multimolecular potential procapsid precursors that are produced during assembly of procapsids """"""""in vivo"""""""" and """"""""in vitro""""""""; for detection and isolation, procedures of gel electrophoresis, density gradient electrophoresis and centrifugation, several recently developed in the laboratory of the PI, will be used, (c) characterize the multimolecular particles isolated using the above procedures and also our newly-developed procedures of gel sieving, (d) determine which of the multimolecular particles isolated are in the assembly pathway of the procapsid and determine the temporal sequence of these particles in the pathway; procedures of kinetic labeling-gel electrophoresis-autoradiography will be used whenever possible, (e) continue our development of gel sieving for determining the shape of a multimolecular particle. The information obtained will assist in understanding mechanisms of the assembly of multimolecular complexes in normal and diseased cells. Some of the procedures developed should have use in diagnostic virology.
| Easom, R A; DeBuysere, M S; Olson, M S et al. (1989) Size determination of multienzyme complexes using two-dimensional agarose gel electrophoresis. Proteins 5:224-32 |
| Serwer, P (1987) The mechanism for producing two symmetries at the head-tail junction of bacteriophages: a hypothesis. J Theor Biol 127:155-61 |
| Serwer, P (1987) Agarose gel electrophoresis of bacteriophages and related particles. J Chromatogr 418:345-57 |
| Serwer, P; Hayes, S J (1986) Exclusion of spheres by agarose gels during agarose gel electrophoresis: dependence on the sphere's radius and the gel's concentration. Anal Biochem 158:72-8 |
| Serwer, P; Hayes, S J; Griess, G A (1986) Determination of a particle's radius by two-dimensional agarose gel electrophoresis. Anal Biochem 152:339-45 |
