Mass analysis of bacteriophage T4 proheads and mature heads by scanning transmission electron microscopy and hydrodynamic measurements.

Quantitative mass analysis of bacteriophage T4 proheads by scanning transmission electron microscopy (STEM) revealed a mass of 79.5 +/- 0.6 MDa, while hydrodynamic measurements yielded a prohead mass of about 80 MDa. This is 25% less than the prohead mass deduced from its polypeptide composition, and this finding implies that the bacteriophage T4 prohead is built of fewer polypeptide copies than previously reported. In contrast, the mass of mature heads measured by STEM, 194 +/- 2 MDa, is in agreement with previous mass measurements of DNA and protein content, and it is consistent with the previously determined stoichiometry. This good agreement of average STEM values for proheads and mature heads with corresponding hydrodynamic measurements suggests that STEM allows faithful evaluation of the masses of large supramolecular assemblies (i.e., greater than or equal to 200 MDa) such as whole viruses or cellular organelles.

Head structure of bacteriophages T2 and T4.

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

Reversible fixation for the study of morphology and macromolecular composition of fragile biological structures.

Many subcellular structures are assemblies of subunits, which are in dynamic equilibrium with free subunits in solution. Their dissociation upon dilution, resulting from cell lysis, can be prevented by adequate fixation. If the latter is reversible, the constituting proteins of such subcellular structures can be analysed electrophoretically. Polyheads of bacteriophage T4 are in dynamic equilibrium with their subunits. They fit very well as a probe to measure the efficiency of crosslinking by the arrest of dissociation upon dilution and upon treatment with hot SDS. Formaldehyde (5%, 20 min, 20 degrees C) leads to a stabilisation comparable to fixation with glutaraldehyde (1%, 30 min). The fixation is shown to be reversible up to 86% by acid and borohydride treatment, but is stable towards heat and SDS-mercaptoethanol. Bands of the reversed proteins are neat and not found in detectably different positions in comparison to controls, when checked by SDS gel electrophoresis.