Abstract
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. 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.
Highlights
Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer
Using Escherichia coli Dihydrofolate reductase (DHFR) it was found that the four common mutations leading to drug resistance proceed in a stepwise fashion and that the second of the four mutations, P21L, involves a residue that is not in one of the active sites, but rather is part of a dynamical loop region that allows access to the binding pocket[3]
The loop motion impacted by the ec DHFR P21L mutation can be associated with long range structural vibrations of the protein backbone that lie in the terahertz (THz) frequency range
Summary
Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. 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 loop motion impacted by the ec DHFR P21L mutation can be associated with long range structural vibrations of the protein backbone that lie in the terahertz (THz) frequency range While these vibrations had long been discussed theoretically, they have only recently been measured experimentally[4,5,6,7]. Anisotropic absorption can provide a more detailed and specific measurement to distinguish those vibrations that impact function This simple picture requires either measuring a single protein, or a sample where all the proteins are aligned. We characterize and model PV-ATM using single crystal sucrose, and demonstrate unique dynamical fingerprinting of bench marking biomolecules CEWL, DHFR, photoactive yellow protein (PYP) and RNA Gquadruplex[20]
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