Gillilan, R. E. | bioRxiv Science

bioRxiv2023

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of di-nitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at non-cryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the Fumarate and Nitrate Reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present, and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR binding sites. By coupling SEC-anSAXS with full spectrum UV-Vis analysis, we show that the [4Fe-4S] clustercontaining dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

biochemistry↗

bioRxiv2022

Inline SAXS-coupled chromatography under extreme hydrostatic pressure

As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. The apparatus described here functions at a range of temperatures (0{degrees} C - 50{degrees} C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

biophysics↗