Novel “dark state” phosphate cluster formation in aqueous solution

Phosphates and polyphosphates play a ubiquitous role in biology, from structural components including cell membranes and bone to energy storage via ATP, but the solution phase space leading to the formation of polyphosphates is not entirely understood. We present novel studies in which the behavior of phosphates with increasing temperature via ³¹P NMR exhibit anomalous relaxation, indicating the presence of a novel phosphate state. Further investigations reveal that these “dark states” that have previously eluded detection to be assemblies of phosphates forming at up to a micron in size at elevated temperatures. The formation of these assemblies appears to be entropically driven, possibly via depletion interactions, producing a liquid-like condensed phase of phosphates, and persisting with a variety of different phosphate-containing species, including adenosine phosphates. We also explore the phase space dictating the formation of these assemblies and the mechanisms mediating their clustering utilizing dynamic light scattering and ³¹P solution NMR techniques. These results suggest that hidden phosphate assemblies can occur under biologically relevant conditions, lending more insight into the interpretation of in vitro and in vivo phosphate containing pathways and species.