Probing nucleotide binding site of annexin A6

A putative consensus site for GTP/ATP binding has been recently identified within human annexin A6 (AnxA6) by using molecular modeling, infrared (IR), and fluorescence spectroscopies. In this report. we describe the structural changes of human recombinant AnxA6 caused by binding of a non-hydrolyzable GTP analog, GTP-gamma-S photoreleased from GTP-gamma-S[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged GTP-gamma-S). Recording the infrared spectra of AnxA6 before and after photorelease of GTP-gamma-S and measuring directly their differences revealed time-dependent structural changes caused by the photorelease of GTP-gamma-S and its binding to AnxA6. The infrared difference spectra consisted of a decrease in the intensity of the 1660-1655-cm(-1) band and a concomitant increase of the 1622-1617-cm(-1) component band. This suggests that carbonyl groups of peptide backbone are affected due to a small distortion of secondary structure of AnxA6 upon nucleotide binding. The infrared spectra measured as function of time, served to generate 2D IR correlation spectra. Our findings indicated that both bands were correlated, suggesting that they belong to the same molecular species. (C) 2004 Elsevier B.V. All rights reserved.