Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure.

During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes. We investigated in detail protein systems with different shape morphologies (bovine serum albumin, microtubules), which have been proposed to be susceptible to THz-radiation, under variable parameters (THz wavelength, THz power densities up to 6.8 mW/cm2, protein concentrations). None of the studied systems and conditions revealed structural changes detectable by SAXS suggesting that the expected non-thermal THz-induced effects do not lead to alterations of the overall structures, which are revealed by scattering from dissolved macromolecules. This leaves us with the conclusion that, if such effects are present, these are either local or outside of the spectrum and power range covered by the present study.

Rapid screening of in cellulo grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach.

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.