Researchers report that they have developed a method for rapidly measuring proteins’ unique vibrations, which help them perform vital tasks ranging from cell repair to photosynthesis. The advance could open new possibilities in biological research, such as studying the microscopic motions of proteins more efficiently, or leveraging vibrational patterns as “fingerprints” to quickly determine whether specific proteins are present in a laboratory sample, according to Andrea Markelz, PhD, a professor of physics at the University of Buffalo (UB) College of Arts and Sciences.
Scientists could also use the new technique to swiftly assess whether pharmaceuticals designed to inhibit a protein’s vibrations are working, she adds. This would require comparing the vibrational signatures of proteins before and after the application of inhibitors.
“Proteins are elegant and robust nanomachines that nature has developed,” says Markelz. “We know nature uses molecular motions to optimize these machines. By learning the underlying principles of this optimization, we can develop new biotechnology for medicine, energy harvesting, and even electronics.”
Katherine A. Niessen, PhD, a UB researcher who is now a development scientist at Corning, is first author of the paper (“Protein and RNA dynamical fingerprinting”), which appears in Nature Communications. Collaborators include scientists in the UB department of physics, the UB department of structural biology in the Jacobs School of Medicine and Biomedical Sciences at UB, the Hauptman-Woodward Medical Research Institute, the National Heart, Lung, and Blood Institute, and the University of Wisconsin-Milwaukee. The work was funded by the National Science Foundation and U.S. Department of Energy.
“Protein structural vibrations impact biology by steering the structure to functional intermediate states, enhancing tunneling events, and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique, however, has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community,” the investigators wrote.
“Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.”
Protein vibrations enable proteins to change shape quickly so they can readily bind to other proteins. Several years ago, Markelz’ lab developed a technique called anisotropic terahertz microscopy (ATM) to observe protein vibrations in detail, including the energy and direction of movements. In ATM, researchers shine terahertz light on a molecule. Then, they measure the frequencies of light the molecule absorbs. This provides insight into the molecules’ motion because molecules vibrate at the same frequency as the light they soak up.
The new study reports that Markelz’ team has improved upon ATM by overcoming one of the method’s limitations: the need to painstakingly rotate and re-center protein samples several times in a microscope to gather enough useful data.
Now, “instead of rotating the protein sample, we rotate the polarization of the light we shine on the sample,” Markelz said. With this adjustment, it takes just four hours to make useful measurements, six times faster than before. The new technique also generates more detailed data.
Using the new approach, Markelz and colleagues measured the vibrations of four different proteins, generating a recognizable vibrational fingerprint for each that consisted of the molecule’s unique light absorption pattern. The proteins studied were chicken egg-white lysozyme, photoactive yellow proteins, dihydrofolate reductase (a drug target for antibiotics and cancer), and RNA G-quadruplexes (thought to be involved in vital cellular functions such as gene expression).
The new method produced distinct light-absorption spectra for chicken egg-white lysozymes that were freely moving versus chicken egg-white lysozymes that were bound by a compound that inhibits the lysozymes’ function, and alters their vibrations. This demonstrates the technique’s utility in quickly identifying the presence of a working inhibitor, says Markelz.