Novel technique shows molecule conformations in live cells

Comprehending how proteins twist, bend, and shape-shift, as they work in cells, is vital to gain insights into diseases and normal biology. However, the lack of better imaging techniques has made the deeper understanding of protein dynamics elusive.

Novel technique shows molecule conformations in live cells
Klaus Hahn, PhD. Image Credit: UNC School of Medicine.

Recently, for the first time, researchers from the UNC School of Medicine came up with a technique that helps the area to make further advancements.

The researchers’ novel “binder-tag” method permits scientists to identify and track proteins that are in a required shape or “conformation,” and to do so in actual time inside living cells. The researchers put forth a process, basically, movies that pursue the active version of a vital signaling protein—a molecule vital for cell growth, in this case. The research was published in the Cell journal.

No one has been able to develop a method that can do, in such a generalizable way, what this method does. So I think it could have a very big impact.”

Klaus Hahn, PhD, Study Co-Senior Author and Ronald G. Thurman Distinguished Professor, Pharmacology, UNC School of Medicine

Klaus Hahn is also director of the UNC-Olympus Imaging Center, at UNC School of Medicine.

The study was a collaboration between Hahn’s laboratory and the laboratory of imaging analysis expert Timothy Elston, Ph.D., professor of pharmacology and co-director of the Computational Medicine Program at the UNC School of Medicine. Both of them are members of the UNC Lineberger Comprehensive Cancer Center.

Filming the very small

The novel technique, similar to all biological imaging techniques, appeals to the basic issue—the incapability of visualizing accurately and precisely most of the molecules at work in living cells employing an ordinary light microscope. The light flows in huge waves that bend around things and they cannot render objects sharply down at the scales where proteins operate.

A specific approach to this issue, particularly when proteins are to be imaged in their normal live-cell habitats, is to tag the targeted proteins with fluorescent beacons. This is because the beacons’ light emissions can be visualized and captured directly with microscopy—for instance, to map the places where a specific protein works in a cell.

FRET (Förster resonant energy transfer), relying upon exotic quantum effects, fixes pairs of such beacons in target proteins so that their light changes as the protein’s conformation changes. This enables a certain amount of research on protein dynamics as they shape-shift inside cells. However, FRET and other such techniques have limitations, like weak fluorescent signals, that highly restrict their utility.

The novel binder-tag technique begins with the introduction of a small molecular “tag” inside a protein being examined. A separate molecule is later employed which attaches to the tag only when the tag-containing protein takes a specific shape or conformation, like when the protein is active to aid a cell to carry out a specific function.

Positioning suitable fluorescent beacons inside the binder and/or the tag molecule efficiently enables a scientist to image, over time, the accurate locations of tagged proteins that are in a specific conformation of interest.

The technique is compatible with a broad range of beacons, including effective ones than the interacting beacon pairs needed for ordinary FRET. According to Hahn, a binder-tag can also be employed to build FRET sensors effortlessly. Furthermore, the binder-tag molecules were selected so that nothing in cells can react with them and interfere with their imaging role.

Hahn says that the overall result is a sophisticated technique that in principle can handle a wide variety of protein-dynamics research works out of reach earlier. This also includes investigations of proteins found scarcely in cells.

Hahn and co-workers, in their article, debated various proof-of-principle demonstrations. They employed the novel process to image a vital growth-signaling protein named Src to disclose, in unparalleled detail, how it develops small islands of activity. Sequentially, this allowed the scientists to examine factors impacting the protein’s biological roles.

With this method, we can see, for example, how microenvironmental differences across a cell affect, often profoundly, what a protein is doing.”

Klaus Hahn, PhD, Study Co-Senior Author and Ronald G. Thurman Distinguished Professor, Pharmacology, UNC School of Medicine

Scientists currently employ this technique to map the dynamics of other vital proteins. They are also carrying out additional analyses to reveal how binder-tag can be tailored to capture the dynamics of various protein structures and functions, not merely proteins that function like Src.

The researchers visualize that binder-tag eventually will become a fundamental enabling method for examining normal proteins, bigger multi-molecular structures in cells, and also the dysfunctional proteins linked to diseases like Alzheimer’s.

For a lot of protein-related diseases, scientists haven’t been able to understand why proteins start to do the wrong thing. The tools for obtaining that understanding just haven’t been available.”

Klaus Hahn, PhD, Study Co-Senior Author and Ronald G. Thurman Distinguished Professor, Pharmacology, UNC School of Medicine

Source:
Journal reference:

Liu, B., et al. (2021) Biosensors based on peptide exposure show single molecule conformations in live cells. Cell. doi.org/10.1016/j.cell.2021.09.026.

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