Neal J. Zondlo
University of Delaware
Perfluoro-tert-butyl Amino Acids to Sensitively Probe Protein Function
NMR spectroscopy is limited both by sensitivity and by signal-to-noise. We have synthesized perfluoro-tert-butyl amino acids, ethers of tyrosine, 4R-hydroxyproline, 4S-hydroxyproline, and homoserine, and examined their application to probe protein structure and function. Perfluoro-tert-butyl amino acids have 9 equivalent fluorines, which present as a singlet by 19F NMR. The presentation of 9 fluorines as a singlet renders perfluoro-tert-butyl amino acids particularly sensitive as chemical probes.Within peptides, perfluoro-tert-butyl amino acids may be detected by simple pulse sequences in 30 seconds at 500 nM concentration, appropriate for the interrogation of function at biologically relevant concentrations. Perfluoro-tert-butyl tyrosine exhibits particularly sharp linewidths. Perfluoro-tert-butyl homoserine was incorporated at the site of a recognition leucine, yielding similar affinity in a coactivator peptide-estrogen receptor protein-protein interaction and allowing quantification of complex affinity by 19F NMR spectroscopy. 4R- and 4S-perfluoro-tert-butyl hydroxyprolines exhibit distinct conformational preferences. 4R- and 4S-perfluoro-tert-butyl hydroxyprolines were incorporated in peptides with optimized recognition sequences for the protein kinases PKA and Akt.
Phosphorylation of these peptides resulted in 19F chemical shift changes, allowing the quantification of protein kinase activity by 19F NMR in solution and in HeLa cell extracts. The distinct conformations of these amino acids were differentially recognized by the enzymes, demonstrating conformational selection by protein kinases. In addition, 4,4-difluoroproline was demonstrated to be an exquisite probe of proline conformation, allowing the development of new understanding of the mechanism of polyproline I/polyproline II helix interconversion, the determinants of stability of the polyproline I helix, and bases for the stabilization of cis amide bonds.