PNAS never reported a point: the strange way phosphates interact with water

Researchers at the University of California, Santa Barbara and New York University (NYU) made a startling discovery when they directly investigated the mechanism of assembly of calcium-phosphate clusters: phosphate ions in water have a strange habit of spontaneously alternating
between common hydration states and mysterious previously unreported "dark" states.
This recently discovered behavior, they say, has implications
for understanding the role of phosphates in biocatalysis, cellular energy balance and biomaterial formation.
Their findings were published in the Proceedings of the National Academy of
Sciences.

"Phosphates are everywhere," said Songi Han, a professor of chemistry at UCSB and one of the authors of a paper in the Proceedings of the National Academy of
Sciences.
The ions are surrounded by one phosphorus atom and four oxygen atoms
.
"It's in our blood and serum," Han continued
.
"It's in every biologist's buffer, it's on
our DNA and RNA.
" It is also a structural component of
our bones and cell membranes.

When combined with calcium, phosphate forms small clusters
of molecules during the formation of mineral deposits in cells and bones.
That's what Han and UCSB collaborators Matthew Helgeson and New York University's Alexej Jerschow are preparing to study and describe, hoping to reveal quantum behavior
in symmetric phosphate clusters proposed by UCSB physics professor Matthew Fisher.
But first, the researchers had to set up controlled experiments that included scanning calcium deficient phosphate ions
by nuclear magnetic resonance (NMR) spectroscopy and cryogenic transmission electron microscopy.

However, because students at UCSB and NYU are collecting reference data involving the isotope phosphorus-31, which occurs naturally in aqueous solutions at different concentrations and temperatures, their results did not match
expectations.
For example, Han said, this line represents that^P31 during a spectral NMR scan should narrow
as temperature increases.

"The reason is that the higher the temperature, the faster the molecules tumble," she explains
.
Typically, this rapid molecular motion averages out anisotropic interactions, or interactions that depend on the relative orientation of these small molecules
.
The result will be a narrowing
of the resonance measured by the NMR instrument.

"What we're looking forward to is a phosphorus NMR signal, which is a simple signal where peaks shrink
as temperature increases," she said.
Surprisingly, however, the spectrum we measured is getting wider, the exact opposite
of what we expected.
”

This counterintuitive result set the team down a new path, conducting experiment after experiment to determine the cause
at its molecular level.
After ruling out one hypothesis after another, what is the conclusion? Phosphate ions form clusters under a wide range of biological conditions that evade direct spectral detection, which may be why
they have not been observed before.
In addition, the measurements showed that the ions alternated between the visible "free" state and the dark "combined" state, so the signal became wider rather than a sharp peak
.

In addition, according to co-lead author Mesopotamia Nowotarski, the number of these assembly states increases as the temperature increases, another temperature-dependent behavior
.

"The conclusion from these experiments is that phosphates are dehydrating, which allows them to get closer
," she said.
At lower temperatures, the vast majority of phosphates in solution attach to water molecules, which form a protective water jacket
around them.
This hydration state
is often assumed when considering the behavior of phosphates in biological systems.
But at higher temperatures, Nowotarski explains, they shed their water shields, allowing them to stick to
each other.
This concept was confirmed by NMR experiments probing the phosphate water crust and further validated by analyzing cryogenic transmission electron microscopy images to determine the presence of clusters, as well as co-lead author Joshua Straub's energetic modeling
of phosphate assembly.

According to the researchers, these dynamic phosphate combinations and hydrated shells have important biological and biochemical implications
.
Matthew Helgeson, a chemical engineer, said phosphate is a commonly understood "currency" that stores and consumes energy
in biological systems by converting to adenosine triphosphate (ATP) and adenosine diphosphate (ADP).
"If phosphate hydrate, ADP and ATP represent small 'paper money,' this new finding suggests that these small currencies can be exchanged for much larger denominations — say, $100 — which may interact with biochemical processes very differently
from currently known mechanisms," he said.

In addition, many biomolecular components include phosphate groups, which may similarly form clusters
.
So the discovery that these phosphates can assemble spontaneously may provide some clues to other fundamental biological processes, such as biomineralization — how shells and bones form, and protein interactions
.

"We also tested a range of phosphates, including those incorporated into the ATP molecule, which all exhibited the same phenomenon, and we achieved a quantitative analysis of these combinations," said co-lead author Jiaqi Lu
.

This once-overlooked process may also be important in cell signaling, metabolism, and disease processes, such as Alzheimer's disease, where a phosphate group or phosphorylation attaches to tau proteins in our brains, often found in nerve fiber tangles — hallmarks
of neurodegenerative diseases.
After observing and studying this assembly behavior, the team is now digging deeper to investigate the effects of pH on phosphate assembly, genetic translation, and modified protein assembly, as well as their original work
on calcium phosphate assembly.

"This really changes the way we think about the action of phosphate groups, which we don't normally think of as a driver of molecular assembly," Han said
.

The authors also include Tanvi Sheth and Sally Jiao
of the University of California, Santa Barbara.