We have developed a new class of biocomposites from the non-enzymatic reaction of low complexity protein domains with energy-rich polyphosphate (polyP). The non-enzymatic, post-translational modification of selected protein domains has only recently been reported and is termed polyphosphorylation. We have used gel electrophoresis and ³¹P NMR to measure the reactivity of proteins containing the polyacidic serine and lysine-rich clusters (PASK domains) that spontaneously react with polyP to create covalently-linked organic-inorganic composites. We have found that the PASK domains identified in topoisomerase I and RTS1 are portable and maintain their reactivity with polyP when incorporated into protein constructs that can be self-assembled into larger ordered structures. Understanding the reaction mechanism is critical for the design of supramolecular biocomposites, and we have measured the reactivity in protein constructs containing the PASK domain as a function of the protein sequence, reagent concentration, polyP molecular weight, and the presence of additives. A variety of reaction products are possible from the reaction of one or more lysines in the PASK domains and the structure of the reaction products has been investigated by 1D, 2D ¹H and ³¹P NMR, and the diffusion coefficients of the reactants and products have been measured by pulse-field gradient NMR. The results show that new peaks in the ³¹P NMR spectra result from a phosphonamidite linkage between polyP and the PASK domain.

The TopoI-4E-BP1 construct in the absence (-) and presence (+) of polyP.

Patrick Dennis

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