A recent discovery in the field of Earth science where researchers have found evidence of a “hidden state” related to one of the most abundant ions on the planet. However, without more context, it is not possible to determine what specifically the “hidden state” is or which ion it involves.
While conducting an otherwise straightforward investigation into the assembly mechanism of calcium-phosphate clusters, researchers at UC Santa Barbara and New York University (NYU) made a surprising discovery: In water, phosphate ions have the peculiar tendency of spontaneously switching between their usual hydrated form and an enigmatic, unexplained “black” state.
They claim that this freshly discovered behavior has consequences for comprehending how phosphate species function in biocatalysis, cellular energy balance, and the synthesis of biomaterials. Their findings are published in the Proceedings of the National Academy of Sciences.
“Phosphate is everywhere,” said UCSB chemistry professor Songi Han, one of the authors of a paper in the Proceedings of the National Academy of Sciences. The ion consists of one phosphorus atom surrounded by four oxygen atoms. “It’s in our blood and in our serum,” Han continued. “It’s in every biologist’s buffer, it’s on our DNA and RNA.” It’s also a structural component of our bones and cell membranes, she added.
Phosphates join with calcium to form tiny molecule clusters that eventually become mineral deposits in bone and cells. That’s what Han and collaborators Matthew Helgeson at UCSB and Alexej Jerschow at NYU were preparing to study and characterize, in hopes of uncovering quantum behaviors in symmetric phosphate clusters proposed by UCSB physics professor Matthew Fisher.
But first, the researchers had to set up control experiments, which involved scans of phosphate ions in the absence of calcium via nuclear magnetic resonance (NMR) spectroscopy and cryogenic transmission electron microscopy (cryo-TEM).
The project’s UCSB and NYU students were gathering reference data using the naturally occurring isotope phosphorous 31 in aqueous solutions at various concentrations and temperatures, but their findings didn’t match up with what was anticipated.
Phosphate is everywhere. The ion consists of one phosphorus atom surrounded by four oxygen atoms. It’s in our blood and in our serum. It’s in every biologist’s buffer, it’s on our DNA and RNA.
Professor Songi Han
For instance, Han said, “the line that represents the spectrum for 31P during NMR scans is supposed to narrow with increasing temperatures.”
“The reason is, as you go to higher temperatures, the molecules tumble faster,” she explained.
Typically, this rapid molecular motion would average out the anisotropic interactions, or interactions that are dependent on the relative orientations of these small molecules. The result would be a narrowing of resonances measured by the NMR instrument.
“We were expecting a phosphorus NMR signal, which is a simple one, with a peak that narrows with higher temperatures,” she said. “Surprisingly, though, we measured spectra that were broadening, doing the complete opposite of what we expected.”
The researchers embarked on a new course after this unexpected result, conducting experiment after experiment to pinpoint the culprit at the molecular level. The conclusion, after a year of eliminating one hypothesis after another?
The reason they had not been noticed before is probably because phosphate ions were forming clusters under a variety of biological circumstances, clusters that were eluding direct spectroscopic detection.
Furthermore, the measurements suggested these ions were alternating between a visible “free” state and a dark “assembled” state, hence the broadening of the signal instead of a sharp peak.
Furthermore, the number of these constructed states also increased with temperature, another temperature-dependent characteristic, according to co-lead author Mesopotamia Nowotarski.
“The conclusion from those experiments was that the phosphates are dehydrating and that allows them to come closer together,” she said.
Most of these phosphates in solution adhere to water molecules at lower temperatures, forming a protective water shell around them. In order to understand how phosphorus behaves in biological systems, this hydrated condition is often assumed.
But Nowotarski noted that at greater temperatures, they lose their water barriers, allowing them to adhere to one another. This concept was confirmed by NMR experiments that probed the phosphate water shell, and further validated by analysis of cryo-TEM images to identify the existence of clusters, as well as modeling the energetics of phosphate assembly by co-lead author Joshua Straub.
The researchers claim that these dynamic phosphate assembly and hydration shells have significant ramifications for biology and biochemistry. According to chemical engineer Matthew Helgeson, phosphate is a commonly recognized “currency” that biological systems employ to store and expend energy by converting it into adenosine triphosphate (ATP) and adenosine diphosphate (ADP).
“If hydrated phosphate, ADP and ATP represent small ‘bills’ of currency, this new discovery suggests that these smaller currencies can exchange with much larger denominations say $100 which may have very different interactions with biochemical processes than currently known mechanisms,” he said.
Phosphate groups, which may also organize into clusters, are present in a large number of biomolecular components. Therefore, the discovery that these phosphates can spontaneously assemble may help us understand several other basic biological processes, including protein interactions and biomineralization, which is the process by which shells and skeletons are formed in living things.
“We also tested a range of phosphates, including those incorporated into the ATP molecule, and they all appear to show the same phenomenon, and we achieved quantitative analysis for these assemblies,” said co-lead author Jiaqi Lu.
The attachment of a phosphate group, or phosphorylation, to the protein tau in human brains, which is frequently found in neurofibrillary tangles, a hallmark of neurodegeneration, may be important in the areas of cell communication, metabolism, and disease processes, such as Alzheimer’s disease.
After observing and studying this assembly behavior, the team is now doing more research. In addition to their original work on calcium phosphate assembly, they are looking at the impact of pH on phosphate assembly, genetic translation, and changed protein assembly.
“It really changes the way we think about the role of phosphate groups that we typically don’t consider a driver of molecular assembly,” Han said.
Research in this paper was also conducted by Tanvi Sheth and Sally Jiao at UC Santa Barbara.
