Probing the Molecular Architecture and Assembly of Synthetic and Fungal Melanins with Solid-State NMR

Abstract

Melanins are ubiquitous natural pigments that are associated with biological roles in protection against sunlight, camouflage, free radical scavenging, and microbial virulence. However, elucidating structural characteristics of these enigmatic pigments remains a challenging task because of their insolubility and heterogeneous properties. High-resolution solid-state NMR spectroscopy provides a unique tool to unravel the mysteries of melanin structure, and melanogenesis in Cryptococcus neoformans (CN) requiring exogenous obligatory catecholamine precursors such as L-dopa offers unprecedented opportunities to probe melanin biosynthesis and assembly in fungal cells. 1D solid-state 13C NMR has been used to assess the structural differences among CN melanins produced by systematic variation of catecholamine precursors and in cell-free media. The precursor preferences of CN melanogenesis have been demonstrated by competition studies in which the cells were provided with equimolar mixtures of catecholamines, and a mechanistic scheme adapted from the Mason-Raper pathway has been proposed to rationalize the possible intermediates for biosynthesis of indole-based melanin polymers. 13C and 15N solid-state NMR, including 2D through-space and through-bond correlation spectra, have been implemented to elucidate the ultimate molecular framework of the pigments and melanization in CN with exogenously 13C-enriched L-dopa and/or [U-13C6]-glucose supplied to the growth media. Findings include: (1) the main aromatic components are located spatially within 5-7 A of the long-chain aliphatics; (2) glucose scrambles metabolically to appear in oxygenated- and long-chain aliphatic structures; (3) some aromatic, oxygenated- and long-chain aliphatic moieties are attached covalently to carboxyl groups of the pigment; (4) the pigment is likely to be covalently bound to the polysaccharides of fungal cell wall. The molecular architecture of natural melanin revealed by the solid-state NMR study can guide the design of new drugs against microbial virulence and novel therapeutics for melanoma tumors.