Dynamic Nuclear Polarization Enhanced Multiple-Quantum Spin Counting of Molecular Assemblies in Vitrified Solutions.

Crystallization pathways are essential to various industrial, geological, and biological processes. In nonclassical nucleation theory, prenucleation clusters (PNCs) form, aggregate, and crystallize to produce higher order assemblies. Microscopy and X-ray techniques have limited utility for PNC analysis due to the small size (0.5-3 nm) and time stability constraints. We present a new approach for analyzing PNC formation based on 31P nuclear magnetic resonance (NMR) spin counting of vitrified molecular assemblies. The use of glassing agents ensures that vitrification generates amorphous aqueous samples and offers conditions for performing dynamic nuclear polarization (DNP)-amplified NMR spectroscopy. We demonstrate that molecular adenosine triphosphate along with crystalline, amorphous, and clustered calcium phosphate materials formed via a nonclassical growth pathway can be differentiated from one another by the number of dipolar coupled 31P spins. We also present an innovative approach for examining spin counting data, demonstrating that a knowledge-based fitting of integer multiples of cosine wave functions, instead of the traditional Fourier transform, provides a more physically meaningful retrieval of the existing frequencies. This is the first report of multiquantum spin counting of assemblies formed in solution as captured under vitrified DNP conditions, which can be useful for future analysis of PNCs and other aqueous molecular clusters.

Spectroscopically dark phosphate features revealed by chemical exchange saturation transfer.

Phosphate is an essential anion in the human body, comprising approximately 1% of the total body weight, and playing a vital role in metabolism, cell membranes, and bone formation. We have recently provided spectroscopic, microscopic, and computational evidence indicating that phosphates can aggregate much more readily in solution than previously thought. This prior work provided indirect evidence through the observation of unusual 31 P NMR relaxation and line-broadening effects with increasing temperature. Here, we show that, under conditions of slow exchange and selective RF saturation, additional features become visible in chemical exchange saturation transfer (CEST) experiments, which appear to be related to the previously reported phosphate clustering. In particular, CEST shows pronounced dips several ppm upfield of the main phosphate resonance at low temperatures, while direct 31 P spectroscopy does not produce any signals in that range. We study the pH dependence of these new spectroscopic features and present exchange and spectroscopic parameters based on fitting the CEST data. These findings could be of importance in the investigation of phosphate dynamics, especially in the biological milieu.

Lithium isotopes differentially modify mitochondrial amorphous calcium phosphate cluster size distribution and calcium capacity.

Lithium is commonly prescribed as a mood stabilizer in a variety of mental health conditions, yet its molecular mode of action is incompletely understood. Many cellular events associated with lithium appear tied to mitochondrial function. Further, recent evidence suggests that lithium bioactivities are isotope specific. Here we focus on lithium effects related to mitochondrial calcium handling. Lithium protected against calcium-induced permeability transition and decreased the calcium capacity of liver mitochondria at a clinically relevant concentration. In contrast, brain mitochondrial calcium capacity was increased by lithium. Surprisingly, 7Li acted more potently than 6Li on calcium capacity, yet 6Li was more effective at delaying permeability transition. The size distribution of amorphous calcium phosphate colloids formed in vitro was differentially affected by lithium isotopes, providing a mechanistic basis for the observed isotope specific effects on mitochondrial calcium handling. This work highlights a need to better understand how mitochondrial calcium stores are structurally regulated and provides key considerations for future formulations of lithium-based therapeutics.

Phosphates form spectroscopically dark state assemblies in common aqueous solutions.

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.

Adenylate kinase derived ATP shapes respiration and calcium storage of isolated mitochondria.

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state and thus a prominent analyte in metabolism research. Beyond adenylate interconversion via oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to successfully predict their abundance in an experimental system and to correctly interpret many aspects of mitochondrial function. We showcase here how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Parameters affected include cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics. Study of the latter revealed that the presence of ATP is required for mitochondrial calcium to be shaped into a particularly dense form of mitochondrial amorphous calcium phosphate.