Characterising Side Chain Conformations in Proteins Using ¹⁹F-ENDOR and New Fluorinated Amino Acid Spin Labels

It has recently been shown that distances > 20 Å can be measured between Gd³⁺ spin tags and ¹⁹F labels in proteins using ¹⁹F-ENDOR at 94 GHz. Here we examine the precision with which this method can locate the positions of (aromatic) amino acid side chains and, with the help of established simulation tools, determine the conformational space sampled by solvent-exposed chains in solution. First, using a novel set of fluorinated phenylalanine amino acids incorporated into the metalloprotein Calbindin D_9k binding Gd³⁺, we show that triangulation of the corresponding ¹⁹F-ENDOR determined distances can precisely identify the conformation of buried side chains. The obtained conformation agrees with the same (single) orientation seen in the crystal structure of the native protein. In a second set of proteins labeled with a Gd³⁺ spin tag and noncanonical ¹⁹F-labeled amino acids, the splittings, lineshapes, and integrated intensities of the ¹⁹F-ENDOR signals were used to constrain the conformational space of the side chains initially identified by comprehensive rotamer simulations. This work provides the first example where ¹⁹F-ENDOR constraints have been used to accurately pinpoint side chain conformations in a protein with the help of differently fluorinated amino acids.