Phosphates form spectroscopically dark state assemblies in common aqueous solutions

Abstract

Phosphates and polyphosphates play ubiquitous roles in biology as integral structural components of cell membranes and bone, or as vehicles of energy storage via adenosine triphosphate and phosphocreatine. The solution phase space of phosphate species appears more complex than previously known. We present nuclear magnetic resonance (NMR) and cryogenic transmission electron microscopy (cryo-TEM) experiments that suggest phosphate species including orthophosphates, pyrophosphates, and adenosine phosphates associate into dynamic assemblies in dilute solutions that are spectroscopically "dark." Cryo-TEM provides visual evidence of the formation of spherical assemblies tens of nanometers in size, while NMR indicates that a majority population of phosphates remain as unassociated ions in exchange with spectroscopically invisible assemblies. The formation of these assemblies is reversibly and entropically driven by the partial dehydration of phosphate groups, as verified by diffusion-ordered spectroscopy (DOSY), indicating a thermodynamic state of assembly held together by multivalent interactions between the phosphates. Molecular dynamics simulations further corroborate that orthophosphates readily cluster in aqueous solutions. This study presents the surprising discovery that phosphate-containing molecules, ubiquitously present in the biological milieu, can readily form dynamic assemblies under a wide range of commonly used solution conditions, highlighting a hitherto unreported property of phosphate's native state in biological solutions.

Keywords: assembly; dark state; dehydration; phosphate.

Fig. 1.

³¹P NMR results for phosphate-containing species. (A) 1D NMR spectra from 10 mM sample in (D) taken at every 10 K showing line broadening in orthophosphate. (B) Linewidths for orthophosphate, pyrophosphate, ADP, and ATP as a function of temperature showing monotonic increase with temperature. Solid lines are quadratic fits to data to guide the eye. (C) R₁ and R₂ curves as a function of molecular tumbling rate from Bloembergen–Purcell–Pound theory. Cartoons illustrate the approximate locations of ionic phosphate, ADP, and a standard protein based on tumbling rates. (D) R₂ as extracted from a CPMG pulse sequence and from FWHM for 10 mM and 100 mM monobasic sodium orthophosphate pH 4.5 as a function of temperature, showing monotonic increase in R₂ in each case. Solid lines are quadratic fits to data to guide the eye. R₁ for 10 mM, 100 mM, monobasic sodium orthophosphate pH 4.5 as a function of temperature showing different curve shapes as a function of concentration. Solid lines are cubic fits to data to guide the eye.

Fig. 2.

³¹P CEST results for 100 mM orthophosphate (pH = 4.5). (A) CEST pulse sequence. (B) The experimental and simulated CEST Z-spectra as a function of temperature with 150 Hz of the irradiation pulse power. (C) The width at half height of both the experimental and simulated Z-spectrum dips from (B) as a function of temperature. The solid and dashed lines represent a quadratic fit to data as a guide for the eye.

Fig. 3.

Evidence of phosphate assemblies from TEM and MD simulations. (A and B) TEM images of phosphate assemblies (yellow arrows) after heating phosphate solutions show droplet-like features forming at 25 to 50 nm in size. Samples were from different sources and prepared on different days. (A) 100 mM potassium ADP heated to 343 K before vitrification. (B) 100 mM sodium ADP heated to 343 K before vitrification. (C) Cluster size distributions from MD simulations at 343 K show the fraction, P(N), of phosphate ions in a cluster of size N_clust. The insets show snapshots of phosphate assemblies (red and white) and sodium ions (blue) from the simulations. The cluster size distribution and snapshots show that HPO₄²⁻ strongly assembles in contrast to H₂PO₄⁻. When H₂PO₄⁻ is mixed with HPO₄²⁻, the latter induces clustering of H₂PO₄⁻. In this mixed system, the HPO₄²⁻ ions are grayed out to highlight the clustering of H₂PO₄⁻. Simulation snapshots are visualized using Visual Molecular Dynamics (34).

Fig. 4.

Evidence of entropically driven assembly. (A) 100 mM sodium phosphate at pH 4.2 ³¹P DOSY fits of Log(ψ) vs. gradient strength squared show a linear relationship, indicating that a single diffusion coefficient can describe behavior of phosphorous entities contributing to the observed NMR line. (B) Hydrodynamic diameters extracted from the diffusion coefficient from fits in (A). (C) R₂ and R₁ for potassium phosphate pH 4.5 in the presence of 6k MW polyethylene glycol (PEG) at varying PEG concentrations. Solid lines are quadratic fits to data to guide the eye. (D) R₂ and R₁ for orthophosphate pH 4.5 samples at 10 and 100 mM, with varying types of cationic salt chlorides and concentrations (see legend below). Salt-added samples are sodium phosphate salts, while no additional salt samples shown are monophosphate salts with the corresponding potassium or sodium cationic species. Solid lines are third-order polynomial fits to data to guide the eye. The R₂ trends follow the predicted trends for the Hofmeister series, while R₁ shows little difference at 10 mM phosphate concentration but significant differences for different salts at 100 mM.