Protein-Observed Fluorine NMR (PrOF NMR) spectroscopy is an emerging technique for screening and characterizing small-molecule–protein interactions. The choice of which amino acid to label for PrOF NMR can be critical for analysis. Here we report the first use of a protein containing two different fluoroaromatic amino acids for NMR studies. Using the KIX domain of the CBP/p300 as a model system, we examine ligand binding of several small-molecule compounds elaborated from our previous fragment screen and identify a new ligand binding site distinct from those used by native transcription factors. This site was further supported by computational modeling (FTMap and Schrödinger) and 1H,15N HSQC/HMQC NMR spectroscopy. Metabolic labeling with multiple fluorinated amino acids provides useful probes for further studying ligand binding and has led to new insight for allosterically regulating transcription-factor protein interactions with small-molecule ligands.
Bibliographical noteFunding Information:
This project was supported by the University of Minnesota, the NSF-CAREER Award CHE-1352091, the NIH training grant T32-GM08700, and the UMN Interdisciplinary Doctoral Fellowship (C.T.G.) We gratefully thank Prof. Sandor Vajda for helpful discussions on the applications of FTMap.
- NMR spectroscopy
- biomolecular NMR
- protein–protein interactions