Abstract-Near-Field Stationary Sample Terahertz Spectroscopic Polarimetry for Biomolecular Structural Dynamics Determination

 

Yanting Deng, Jeffrey A. McKinney, Deepu K. George*, Katherine A. Niessen, Akansha Sharma,  Andrea G. Markelz, 

https://pubs.acs.org/doi/10.1021/acsphotonics.0c01876#

THz polarimetry on environmentally sensitive and microscopic samples can provide unique insight into underlying mechanisms of complex phenomena. For example, near-field THz anisotropic absorption successfully isolated protein structural vibrations which are connected to biological function. However, to determine how these vibrations impact function requires high throughput measurements of these complex systems, which is challenged by the need for near field detection, sample environmental control and full polarization variation. Stationary sample anisotropic terahertz spectroscopy (SSATS) and near-field stationary sample anisotropic terahertz microscopy (SSATM) have been proposed using synchronous control of THz and electro optic probe polarizations along an iso-response curve. Here we realize these techniques through robust control and calibration of the THz and NIR polarization states. Both methods rapidly measure the linear dichroism in the far field and near field. Validation measurements using standard birefringent sucrose single crystals found the crystal orientation can be determined by scanning the reference polarization and the synchronous pump–probe polarization settings can be optimized to eliminate artifacts. SSATM is then used to determine spectral reproducibility and dehydration effects for a series of chicken egg white lysozyme samples. Reproducible anisotropic absorbance bands are found at about 30, 44, 55, and 62 cm–1. These bands initially sharpen with slow dehydration, similar to the increase in resolution achieved in X-ray crystallographic protein structure determination. The SSATM technique confirms the reliability of anisotropic absorption characterization of protein intramolecular vibrations and opens an avenue for rapid determination of how these long-range dynamics affect biological function.

A protein’s ‘dance steps’ affect its biological function, study shows

• Mitch Jacoby

http://acsmeetings.cenmag.org/a-proteins-dance-steps-affect-its-biological-function-study-shows/

A new microspectroscopy technique can track changes in the overall direction of complex protein vibrations. The method could enable researchers to determine how an enzyme responds when an inhibitor binds to it, for instance, or when the enzyme develops a mutation.

“Global vibrations” can be thought of as intricate dance steps performed by proteins. The new technique provides an unprecedented up-close look at how those dance steps shift when a protein’s conformation changes, a process that underpins important biological functions.

At the American Chemical Society national meeting in San Francisco on Tuesday, physicist Andrea G. Markelz of the University at Buffalo, SUNY, reported that by using her group’s technique, anisotropic terahertz microscopy, she and her coworkers observed something unexpected.

Working with grad student Katherine A. Niessen and others, Markelz found that a protein can undergo large changes in these dance steps even though the overall protein vibrational energy hardly changes at all (Biophys. J. 2017, DOI: 10.1016/j.bpj.2016.12.049). That’s noteworthy, Markelz explained during a session sponsored by the Division of Physical Chemistry, because some researchers have long speculated that changes in the directions of these global vibrations can boost the efficiency of biological functions such as enzymatic activity. But little experimental evidence has been available to support that idea.

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The Buffalo group now knows why. Researchers had assumed that biologically relevant changes in vibrations would be accompanied by obvious changes in a protein’s vibrational energy states. Because researchers—until now–have been able to only measure energy state distributions, and studies had shown that those didn’t really change, the scientists concluded that the dance steps didn’t change either.

The Buffalo researchers carried out several analyses and found out that those assumptions don’t hold true. First, they used the terahertz microscopy method to study the vibrations of chicken egg-white lysozyme, a natural antibiotic. They compared the free form of the enzyme to the enzyme bound to tri-N-acetyl-D-glucosamine, a compound that inhibits enzyme action. The dance steps of the two forms differed dramatically. Yet inelastic neutron-scattering measurements showed almost no difference in energy.

The team also compared regular lysozyme to a structurally altered form known as a double-deletion mutant. The mutations are located far from the enzyme’s catalytic site and would be expected to have no effect on enzyme action. Yet the mutant mediates catalytic reactions nearly 1.5 times as efficiently as the regular enzyme. The team’s analyses show that the vibrational energy distributions of the two forms are identical. The directions of the vibrations, however, differ markedly, indicating that the mutant’s distinct motions give it a catalytic advantage.

“This is absolutely superb work,” said Steven D. Schwartz, a specialist in theoretical and computational biochemistry at the University of Arizona. Schwartz explained that there is an important ongoing debate regarding the role global vibrations play in the catalytic functions of enzymes. Settling that debate requires direct measurement of these motions while enzymes are carrying out reactions. “That is precisely the promise of this of this work,” he said.