Perfluoro-tert-butyl Homoserine is a Helix-Promoting Highly Fluorinated NMR-Sensitive Aliphatic Amino Acid: Detection of the Estrogen Receptor•Coactivator Protein-Protein Interaction by 19F NMR

摘要

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by ¹⁹F NMR and the absence of native ¹⁹F signals in biological contexts. However, the potential applications of ¹⁹F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has 9 equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) co-activator peptides. A peptide containing perfluoro-tert-butyl homoserine at the i+3 position of the ER coactivator LXX>LL motif exhibited Kd = 2.2 μM for the estradiol-bound estrogen receptor, similar to that of the native ligand. ¹⁹F NMR spectroscopy demonstrated the sensitive detection (5 μM concentration, 128 scans) of peptide binding to ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine to probe protein function and protein-protein interfaces in complex solutions.