Abstract
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.
Highlights
Proteins are key operating elements of complex biological systems, such as cells
This conservation in thermodynamic stability regarding wild type behaviour could be confirmed by monitoring the thermal denaturation of fluorine-labelled BsCspB using one-dimensional 1H and 19F NMR spectroscopy
A combination of CD and NMR spectroscopy showed that the three-dimensional structure and thermodynamic stability of GB1 protein is not significantly affected when 5-19F-Trp has been used for labelling[47]
Summary
Proteins are key operating elements of complex biological systems, such as cells. These macromolecules control a multiplicity of chemical processes and are a central characteristic of living systems[1]. The outstanding question in the application of 19F-NMR based methodologies is how much does the 19F-modification impact the inherent properties of the protein under study, its atomic three-dimensional structure, conformational dynamics, and its overall thermodynamic stability Addressing this question is of high importance as the fluorine-labelled protein variant, and not the wild type, is used in in cellula NMR spectroscopy to report on the native structural and dynamical features of proteins in vivo. In this context, it has been shown that extensively fluorinated amino acids can be effective in increasing protein stability[28] due to the increase in buried hydrophobic surface area as identified in structures solved by X-ray crystallography[29]. The present study closes an important gap in the basic characterization of fluorine-labelled proteins and underlines that this methodology may serve as an optimal tool to study proteins in their native complex biological environment applying high-resolution NMR spectroscopy
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