Simultaneous measurement of multiple fluorine labelling effect on GB1 stability by ¹⁹F NMR

The incorporation of fluorinated amino acids into proteins through natural biosynthesis in E. coli often leads to the production of heterogeneous fluorinated proteins. The stabilities of proteins with different ¹⁹F labelling states can vary, but these differences are challenging to measure due to the difficulty in separating the fluorinated protein mixtures that differ by only a few ¹⁹F atoms. Here, we simultaneously incorporated both fluoro-phenylalanines (3-fluoro-phenylalanine, 3FF; or 4-fluoro-phenylalanine, 4FF) and 5-fluoro-tryptophan (5FW) into GB1 protein. We are able to measure the stability of GB1 protein with different ¹⁹F labelling states without the need for sample separation by taking the advantage of ¹⁹F NMR. The results showed that 4FF-5FW-GB1 with varying ¹⁹F labelling states exhibited significantly different protein stability, with higher 4FF labeling efficiency correlating with decreased stability. Furthermore, residues F₃₀ and F₅₂ show synergistic effects on GB1 stability. In contrast, the 3FF and 5FW substitution exhibits a slightly stabilizing effect on GB1 stability. The present research provides a convenient ¹⁹F NMR method to simultaneously measure fluorine labelling effects on protein stability, favouring precise understanding and analysis of fluorine labelling effects.