A Protein Expression System for Evaluating Cation-PI And PI-PI Interaction in Proteins

Abstract

In proteins phenylalanine, tyrosine, and tryptophan can be involved in cation-pi and pi-pi interactions. The importance of these interactions for protein structure and functioning is well recognized but measuring their strength experimentally is problematic. The chemical aminoacylation strategy combined with the Xenopus oocyte expression system was developed by the Dougherty group at Caltech for the production of proteins labeled with fluorinated aromatic amino acids involved in cation-pi interactions. This elegant expression system only yields ng quantities of proteins, limiting the scope to channel-forming proteins analyzed via patch-clamp. We developed a protein expression platform for the evaluation of pi-pi and cation-pi interactions involving tryptophan. This Lactococcus lactis based expression system can produce mg amounts of a set of 6 proteins, each carrying a different electron density in the tryptophan side chain. It was used to express variants of the multidrug repressor protein LmrR. LmrR regulates the drug-induced expression of the ABC type multidrug efflux transporter LmrCD. LmrR was crystallized in its drug-free state and in complex with aromatic substrates showing each substrate is sandwiched between two tryptophan residues, suggesting pi-pi interactions are operative. The importance of pi-pi interactions for LmrR to bind aromatic substrates could now be quantitated using fluorescence, surface plasmon resonance, and X-ray crystallography. In conclusion, the newly developed expression system makes possible a systematic approach to study cation-pi and pi-pi interactions in proteins using a wide range of biophysical methods.