Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein. We show that single fluorine labelling of phenylalanine or tryptophan residues has neither significant impact on thermodynamic stability nor on folding kinetics compared to wild type BsCspB. Structure determination of fluorinated phenylalanine and tryptophan labelled BsCspB using X-ray crystallography reveals no displacements even for the orientation of fluorinated aromatic side chains in comparison to wild type BsCspB. Hence we propose that single fluorinated phenylalanine and tryptophan residues used for protein labelling may serve as ideal probes to reliably characterize inherent features of proteins that are present in a highly biological context like the cell.

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In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study, and has for this reason to be considered. However, most protein studies neglect the presence of these macromolecules. Consequently, we probe here the overall thermodynamic stability of cold shock protein B from Bacillus subtilis (BsCspB) in cell lysate. We found that an increase in cell lysate concentration causes a monotonic increase in the thermodynamic stability of BsCspB. This result strongly underlines the importance of considering the biological environment when inherent protein parameters are quantitatively determined. Moreover, we demonstrate that targeted application of 19 F NMR spectroscopy operates as an ideal tool for protein studies performed in complex cellular surroundings.

A series of mononuclear σ-phenyl ruthenium complexes Ru­(CO)­Cl­(C6H4-R)­(P i Pr3)2 (R = OMe, CH3, H, F, CF3) were synthesized and analyzed with respect to their electrochemical and spectroscopic properties. To these ends, cyclic voltammetry, IR, and UV/Vis/NIR spectroelectrochemistry as well as EPR spectroscopy on their one-electron oxidized radical cations were employed. Experimental work is complimented by quantum chemical calculations. Our studies reveal that the σ-phenyl ligand strongly contributes to the HOMO and actively participates in the redox processes. Despite comparatively smaller ligand contributions, the redox potentials, the position of the CO stretch as well as the oxidation induced CO band shifts are more sensitive toward the σ-Hammett parameter of the 4-substituent than for related styryl complexes with the same Ru­(CO)­Cl­(P i Pr3)2 metal coligand platform. The comparatively high spin density/positive charge at the 4-position of the phenyl ligand leads to oxidatively induced dehydrodimerization of the radical cation of the parent phenyl complex Ru­(CO)­Cl­(C6H5)­(P i Pr3)2 (1) to the biphenylene-bridged dinuclear complex [{Ru­(CO)­Cl­(P i Pr3)2}2(μ-C6H4–C6H4-4,4′)] n+ (6 n+ ). The latter was identified in spectroelectrochemical experiments and authenticated by independent preparation of neutral 6 and monitoring of its spectroelectrochemical behavior.

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution 19 F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.