Evaluation of π-π interactions in proteins using Trp analogs
The side chains of aromatic amino acids are often involved in cation-π and π-π interactions. Despite their importance for protein function, our understanding of these interactions is very limited, because they are difficult to study experimentally. One experimental approach is progressive fluorination of the concerned aromatic amino acid, but published protein expression systems yield only very low amounts of labeled protein.
Here, we present a Lactococcus lactis Trp auxotroph-based expression system for the efficient incorporation (≥ 95%) of multi-fluorinated Trp analogs. With this system, mg amounts of labeled proteins can be produced. The expression level was improved by developing an optimized chemically defined medium (CDM), which contains only half the number of compounds used in a typical L. lactis CDM medium. Based on this L. lactis Trp auxotroph expression system and the new CDM, the π-π interactions between the protein LmrR and some of its ligands were investigated.
LmrR is a dimeric transcriptional repressor from L. lactis that can bind an aromatic drug between two Trp residues at the dimer interface. Progressive fluorination of the Trp residues showed that the affinity for the drug gradually decreased, clearly establishing the importance of π-π interactions for high-affinity drug binding. High-resolution structures of the labeled protein-drug complexes showed for the first time a structural view of the progressive fluorination approach. In conclusion, the developed L. lactis Trp expression system has paved the way to study cation-π and π-π interactions using a variety of biophysical techniques.