Abstract
The Fluc ion channel family comprises dimeric membrane proteins functioning to expel excess fluoride (F-) from the cytoplasm of microorganisms to resist the inhibitory effects of this environmental xenobiotic anion. Recent structures of the E. coli Fluc homolog, EC2, bound to engineered “monobody” proteins selected from phage display libraries reveal multiple side-chain contacts at the channel-monobody interface. Two such monobodies, S9 and S12, share a similar interface structure, but their nanomolar-range binding affinities differ by ~15 fold. We focus on the per-residue energetic contributions to the binding affinity of the S12-Fluc complex by introducing point mutations at polar contacts located at the interface, assessing the change in binding affinity using fluorescence anisotropy. Analysis of these results in comparison to previous work on S9 indicates that while the residues contacting the monbody-EC2 interface are similar for both S9 and S12, the terminal hydroxyl of various tyrosine residues appear crucial to the binding of the S12-Fluc complex, while relatively insignificant to S9-Fluc.