Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques

While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. <i>In vitro</i> capsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses. To overcome this barrier, we present a method that utilizes sequential protein expression and in cell assembly of component-specifically labeled viral capsids in amounts suitable for NMR studies. We apply this approach to purify capsids of bacteriophage ϕ6 isotopically labeled on only one of its four constituent protein components, the NTPase P4. Using P4-labeled ϕ6 capsids and the sensitivity enhancement provided by dynamic nuclear polarization, we illustrate the utility of this method to enable ssNMR studies of complex viruses

Using Solid-State NMR To Monitor the Molecular Consequences of <i>Cryptococcus neoformans</i> Melanization with Different Catecholamine Precursors

Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus <i>Cryptococcus neoformans</i> also offers unique opportunities for investigating melanin development. In the current study, pigments produced with l-dopa, methyl-l-dopa, epinephrine, and norepinephrine precursors are compared structurally using ¹³C and ¹H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of <i>C. neoformans</i> melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions