Researchers centered at the University of California, Santa Barbara (UCSB) have presented a new tool to resolve the structure of membrane-embedded and membrane-associating proteins. The tool, a form of NMR relaxometry, is used to measure the water dynamics gradient above and across the lipid bilayer. According to the researchers, this gradient is an intrinsic ruler—it reveals structural information about the topology, immersion depth, and the location of proteins. It even gives information about protein segments residing well away from the membrane surface.

The UCSB researchers described the new tool in a paper published online September 30 in the Proceedings of the National Academy of Sciences (“Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces”). The work came about when the researchers discovered that water on the membrane surface has a very distinct movement pattern: It is slowed down because the water is attracted to the membrane surface across several water layers. The scientists then wondered whether they could use this as an intrinsic ruler to determine how the associating protein is anchored into the membrane.

“It’s very difficult to determine at what depth and in what conformation the protein is associating with the membrane, especially if you’re talking about the interface or even the surface,” said UCSB’s Song-I Han, Dr.rer.nat., head of the Han Research Group and corresponding author of the study. “We found a contrast mechanism—water dynamics—which is distinctly different even above the membrane surface where there is no lipid density. The membrane surface distinctly changes the property of the water layers above it.”

“Membrane proteins can sit deep inside the membrane but also associate at the periphery of the membrane,” explained postdoctoral scholar Chi-Yuan Cheng, Ph.D., the lead author of the study. “This can play a very important role in function, especially for peripheral and interfacial proteins.”

The team used Overhauser dynamic nuclear polarization-enhanced nuclear magnetic resonance (NMR), a technique they developed over the last few years. Using a small and stable radical with an even higher magnetic property than the hydrogen atom of water, researchers exploited this magnetic dipole by inserting it as a spin probe in the protein or membrane position of interest. They then used microwave irradiation to excite the dipole, which subsequently excited nearby water molecules, only when they moved at the same frequency as the dipole. In effect, the water near the spin probe was polarized, causing large NMR signal enhancements that could be measured to extract the local water dynamics.

“People have used this NMR relaxometry method before,” said Dr. Han, “but what is novel is the fact the we use an electron spin as our excitation source, which has a much higher frequency than previously exploited, and that we actively drive the excitation of these spin probes with microwaves. The spin probes process at 10 gigahertz—instead of hundreds of megahertz—which allows us to look at the faster water dynamics relevant here as they are altered on biomolecular surfaces.”

Reflecting on the significance of their work, the study’s authors note that “the structural properties of a membrane-associating protein at the water-membrane interfaces are intimately linked to its biological function, but they are difficult to characterize using existing biophysical tools.” Then, they add that having identified the existence of a distinct intrinsic gradient of water diffusion across the lipid bilayer, which encompasses a thick surface hydration layer above the lipid membrane surface, the gradient may be exploited to characterize otherwise difficult-to-resolve structural details. “This study,” the authors conclude, “demonstrates a potential of a broadly applicable approach for the structure-dynamics-function study of membrane proteins, membrane systems, and beyond.”

Having completed this proof-of-principal study, Dr. Han’s team intends to follow-up by scrutinizing neurodegenerative proteins, specifically tau, which plays a key role in Alzheimer’s disease. The working hypothesis is that at some aggregation stages, tau may exert toxicity as it breaks through the membrane barrier, in part determined by its slowed surface water dynamics.

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