Fourier transform infrared spectroscopic characterization of a photolabileprecursor of glutamate

Recently, it has been demonstrated that Fourier transform infrared spectros copy (FTIR) detects conformational changes in the glutamate receptor ligand -binding domain that are associated with agonist binding. Combined with fla sh photolysis, this observation offers the prospect of following conformati onal changes at individual protein and agonist moieties in parallel and wit h high temporal resolution. Here, we demonstrate that gamma(alpha-carboxy-2 -nitrobenzyl) glutamate (caged glutamate) does not interact,vith the protei n, and that following photolysis with UV light the FTIR difference spectrum indicated changes in the protein tertiary and secondary interactions. Thes e changes were similar to those observed for the protein upon addition of f ree glutamate. Thus, caged glutamate and its photolysis by-products are ine rt in this system, whereas the released glutamate exhibits full activity. D ifference spectra of caged glutamate and of reaction analogs permitted iden tification of and correction for FTIR signals arising from the photolytic r eaction and confirmed that its products are indeed glutamate and 2-nitrosop henyl glyoxalic acid. (C) 2000 Federation of European Biochemical Societies . Published by Elsevier Science B.V. All rights reserved.