Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy

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

“…Figs. 1 and S1) were expressed in E. coli cells by using pET24a CspB and pAR1219 vectors as described previously 32 (DSMZ 12779 strain was used for the three Phe variants whereas the strain CAG 18455 7371 was used for the three Trp variants). Subsequently, an established protocol for protein purification was applied 32 .…”

“…1 and S1) were expressed in E. coli cells by using pET24a CspB and pAR1219 vectors as described previously 32 (DSMZ 12779 strain was used for the three Phe variants whereas the strain CAG 18455 7371 was used for the three Trp variants). Subsequently, an established protocol for protein purification was applied 32 . The concentration, c, of the purified proteins was determined by measuring the absorbance at λ = 280 nm, A 280 , of the protein solution in a d = 1 cm long cuvette in an UV/Vis spectrometer (Agilent 8453 UV-visible Spectroscopy System, Agilent Technologies) employing extinction coefficients of ε°2 80 = 2705 M −1 cm −1 (4-19 F-Trp BsCspB), ε 280 = 2887 M −1 cm −1 (5-19 F-Trp BsCspB), ε 280 = 2575 M −1 cm −1 (6-19 F-Trp BsCspB), and ε 280 = 5800 M −1 cm −1 (2-19 F-Phe BsCspB, [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F-Phe BsCspB, 4-19 F-Phe BsCspB, and wild type BsCspB) 32 .…”

“…Within these residues, we used different sites for the incorporation of the fluorine atoms, generating a total of three Phe and three Trp modified amino acids (Fig. 1) 32 . Using this tool kit, we were then able to successfully prepare six different 19 F-labelled BsCspB variants (2-19 F-Phe-, 3-19 F-Phe-, 4-19 F-Phe-, 4-19 F-Trp-, 5-19 F-Trp-and 6-19 F-Trp-BsCpB) at a milligram scale and in high purity enabling high resolution one-dimensional 1 H, 19 F and two-dimensional 1 H-15 N correlation NMR spectroscopy of the samples mentioned above 32 .…”

“…1) 32 . Using this tool kit, we were then able to successfully prepare six different 19 F-labelled BsCspB variants (2-19 F-Phe-, 3-19 F-Phe-, 4-19 F-Phe-, 4-19 F-Trp-, 5-19 F-Trp-and 6-19 F-Trp-BsCpB) at a milligram scale and in high purity enabling high resolution one-dimensional 1 H, 19 F and two-dimensional 1 H-15 N correlation NMR spectroscopy of the samples mentioned above 32 . BsCspB is a relatively small protein of 67 amino acids length that belongs to the cold shock protein family [33][34][35] .…”

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

“…[17] As another advantage, it has been shown that the site-directed incorporation of fluorinated tryptophan or phenylalanine residues into a target protein exhibits no or only mild effects on the overall thermodynamic stability and folding kinetics of the modified protein compared to the wild type form. [18] In previous work we presented a robust method to incorporate fluorinated tryptophan or phenylalanine into the cold shock protein from Bacillus subtilis, BsCspB, which inherently contains seven phenylalanine (F9, F15, F17, F27, F30, F38, and F49) and one tryptophan residue (W8). Consequently, [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] FÀ TrpÀ BsCspB comprises a single fluorinated site whereas 4- 19 FÀ PheÀ BsCspB comprises seven fluorinated sites.…”