MACROMOLECULAR STRUCTURE

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The structures and mechanisms of assembly of biological macromolecular, complexes, and subcellular structures are studied, primarily by high-resolution electron microscopy and computer image processing. Two projects completed during the past year concern (1) characterization of a major conformational change in a viral capsid protein that occurs when the precursor capsid is transformed into the mature virion; and (2) determination of the molecular weights, oligomeric status, carbohydrate contents, and two-dimensional mass-map of the gp160/gp120 surface glycoprotein of human immunodeficiency virus, type 1 (HIV-1) . 1) After polymerization of bacteriophage T4 precursor capsid is complete, its surface lattice undergoes a radical, cooperative, conformational change that results in an 18% increase in size, and striking morphological, functional, and immunological differences. We have investigated the conformational basis of this event by using laser Raman spectroscopy to determine the respective secondary structures of the precursor and mature states. Upon expansion, the alpha-helix content of the major capsid protein is reduced from 36% to 21%, and its beta-sheet content increases from 33% to 46%. We conclude that the conformational change involves a fundamental re-folding of a substantial portion of the protein. 2) Scanning transmission electron microscopy has been applied to preparations of gp160/gp120 of HIV-1 purified from a recombinant viral expression system. From these data, we have established that the precursor molecule, gp160, is a dimer of 125 kDa subunits, each of which comprises 91 kDa of protein together with, on average, 34 kDa of oligosaccharides. Thus the nominal SDS-PAGE-derived molecular weight of 160 kDa is a major overestimate, presumably on account of its high carbohydrate contents. Gp120 and gp41, the maturation cleavage products of gp160, have monomer masses of 89 kDa and 36 kDa respectively, indicating that gp120 contains virtually all the carbohydrates present on gp160. Moreover, gp160 monomers dimerize primarily through interactions between their gp41 moieties.