Small-angle x-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model.

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (MarR) family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

Upgraded D22 SEC-SANS setup dedicated to the biology community.

Described here is the current status of the upgraded in situ size-exclusion chromatography (SEC) system implemented with the D22 small-angle neutron scattering (SANS) instrument at the Institut Laue-Langevin. Since its initial proof of principle in 2016, this SEC-SANS arrangement has been continuously requested by the user community, leading to the design of an upgraded version. A detailed description of the setup and its control is provided, and a few examples of protein structural investigations are presented, which will highlight the various possibilities and limitations of the setup to optimize experimental success.

Hydrogen-Deuterium exchange kinetics in ß-lactoglobulin (-)-epicatechin complexes studied by FTIR spectroscopy.

Hydrogen-Deuterium exchange kinetics of ß-lactoglobulin and ß-lactoglobulin (-)-epicatechin solutions has been investigated through the analysis of the amide I absorption band at 1650cm-1 in the FTIR spectrum. H-D substitution in NH amides and residues of the protein results in a slight red-shift and in intensity changes of the amide I components: either these effects have been inspected in the framework of the Principal Components Analysis methods. The present analysis allowed to unveil three H-D kinetics at the timescale of the oligomeric fluctuations of the protein. A fast mechanism (lifetime from 5 to 10min) can be ascribed to the dynamics of protein oligomers and aggregates at the scale of the quaternary structure variations, and it is not observed in the complexes ß-lactoglobulin (-)-epicatechin. The other slowest kinetics, whose lifetimes are in the range 1-10h, are here associated to dynamics of high-molecular weight complexes that hamper the proton exchange. The role of (-)-epicatechin as an enhancer of the formation of stable high-molecular weight aggregates from ß-lactoglobulin is also discussed by comparison of the lifetimes at different protein concentrations.