Protein and RNA dynamical fingerprinting.

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.

Moving in the Right Direction: Protein Vibrations Steering Function.

Nearly all protein functions require structural change, such as enzymes clamping onto substrates, and ion channels opening and closing. These motions are a target for possible new therapies; however, the control mechanisms are under debate. Calculations have indicated protein vibrations enable structural change. However, previous measurements found these vibrations only weakly depend on the functional state. By using the novel technique of anisotropic terahertz microscopy, we find that there is a dramatic change to the vibrational directionality with inhibitor binding to lysozyme, whereas the vibrational energy distribution, as measured by neutron inelastic scattering, is only slightly altered. The anisotropic terahertz measurements provide unique access to the directionality of the intramolecular vibrations, and immediately resolve the inconsistency between calculations and previous measurements, which were only sensitive to the energy distribution. The biological importance of the vibrational directions versus the energy distribution is revealed by our calculations comparing wild-type lysozyme with a higher catalytic rate double deletion mutant. The vibrational energy distribution is identical, but the more efficient mutant shows an obvious reorientation of motions. These results show that it is essential to characterize the directionality of motion to understand and control protein dynamics to optimize or inhibit function.

Terahertz optical measurements of correlated motions with possible allosteric function.

A suggested mechanism for allosteric response is the distortion of the energy landscape with agonist binding changing the protein structure's access to functional configurations. Intramolecular vibrations are indicative of the energy landscape and may have trajectories that enable functional conformational change. Here, we discuss the development of an optical method to measure the intramolecular vibrations in proteins, namely, crystal anisotropy terahertz microscopy, and the various approaches which can be used to identify the spectral data with specific structural motions.

Optical measurements of long-range protein vibrations.

Protein biological function depends on structural flexibility and change. From cellular communication through membrane ion channels to oxygen uptake and delivery by haemoglobin, structural changes are critical. It has been suggested that vibrations that extend through the protein play a crucial role in controlling these structural changes. While nature may utilize such long-range vibrations for optimization of biological processes, bench-top characterization of these extended structural motions for engineered biochemistry has been elusive. Here we show the first optical observation of long-range protein vibrational modes. This is achieved by orientation-sensitive terahertz near-field microscopy measurements of chicken egg white lysozyme single crystals. Underdamped modes are found to exist for frequencies >10 cm(-1). The existence of these persisting motions indicates that damping and intermode coupling are weaker than previously assumed. The methodology developed permits protein engineering based on dynamical network optimization.