Solid-State NMR Studies of Two Backbone Conformations at Tyr185 as a Function of Retinal Configurations in the Dark, Light, and Pressure Adapted Bacteriorhodopsins

Abstract

Backbone conformations near tyrosine in bacteriorhodopsin (bR) corresponding to all-trans and 13-cis retinal configurations in the dark, light, and pressure adapted states were investigated for Tyr-X peptide bonds by using double amino acid labeling with [1-13C]Tyr, [15N]X-bR by means of rotational echo double resonance (REDOR) technique. The NMR signals obtained from the difference spectra between REDOR and full echo experiments allowed selectively detect on a tyrosine signals for Tyr185-Pro186, Tyr26-Phe27, and Tyr64-Gly65. Two 13C NMR peaks were observed for REDOR-filtered spectrum of Tyr185 in the dark at ambient temperature. These two signals are attributed to the bR with all-trans and 13-cis retinals. REDOR-filtered spectra for Tyr26 and 64 show singlet lines because they are located far from the retinal. All-trans peak intensity was increased in the light adapted state, while the 13-cis state increased for the pressure-adapted state. These results indicate that REDOR-filtered experiments provide us with valuable information of protein-retinal interaction.