What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

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.

Insights into Protein Stability in Cell Lysate by 19F NMR Spectroscopy

“…Specifically, Δ G 0 was determined for 5‐ 19 F−Trp− Bs CspB and 4‐ 19 F−Phe− Bs CspB to Δ G 0 =7.2±0.2 and 8.5±0.2 kJ/mol ( 1 H dimension) and to Δ G 0 =7.7±0.2 and 8.7±0.2 kJ/mol ( 19 F dimension), respectively. These numbers are in good accordance to Δ G 0 values determined for fluorinated variants of Bs CspB by fluorescence spectroscopy before using also chemical denaturation [18b] …”

“…We demonstrated the high purity of the protein variants with high‐resolution 1 H and 19 F NMR spectroscopy as well as mass spectrometry [18a] . Moreover, it could be shown by applying two dimensional heteronuclear 1 H, 15 N HSQC NMR spectroscopy and X‐ray crystallography that the fluorine label has no significant impact on the three‐dimensional structure of Bs CspB even when the orientation of fluorinated side chains is analyzed [18b] . Bs CspB is a relatively small protein ( M W =7.3 kDa) constituting a β‐barrel fold and interacting to singly stranded DNA and RNA molecules [19] .…”

“… Cartoon representing the fluorinated variant of cold shock protein B from Bacillus subtilis , 4‐ 19 F−Trp− Bs CspB (cartoon mode, colored in gray, PDB ID: 6SZZ [18b] ) present in a highly crowded environment comprising diverse macromolecules found in cell lysate. The main‐ and side‐chain atoms of 4‐ 19 F−tryptophan are highlighted in blue, except for side chain fluorine at ring position four (colored in red).…”

Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement

“…However, in proteins with fluorinated amino acids, structure and stability do not necessarily correlate, as demonstrated by studies on aromatic amino acids [ 123 ]. In fact, the survey of the crystal structures presented in chapter 6 demonstrates that the native backbone fold of the protein can remain unaltered, while associated biochemical assays may show notable differences in the thermodynamic stability of the fluoroproline-containing proteins.…”

“…By performing experiments to achieve the proteome-wide insertion of fluoroprolines into microbial cells, we should be able to answer the question to what extent naturally evolved protein scaffolds and related cellular machineries and systems are suitable for the accommodation and integration of these isosteric building blocks. The adaptation of bacteria to fluorinated tryptophans discussed above is rather the exception, since tryptophan is a rare amino acid (only 20688 codons in the E. coli genome [ 77 ]), and hydrogen-to-fluorine replacement on the indole side-chains represent "atomic mutations", which are known to be well tolerated even in investigated isolated proteins [ 123 – 124 155 ] and proteomes [ 153 – 154 ]. Thus, it should be taken into account that the natural structural scaffolds with hydrocarbon cores have been formed and optimized through billions of years of evolution and may not be suitable to accommodate a large number of fluorine atoms [ 1 , 8 ].…”