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.