High-Temperature Intermediate State of Bacteriorhodopsin Prior to the Premelting Transition of Purple Membrane Revealed by Reactivity with Hydrolysis Reagent Hydroxylamine

摘要

The structural properties of bacteriorhodopsin (bR) over a wide temperature range of 30-80 deg C have been examined through the hydroxylamine reactivity of the Schiff base inside the molecule and through hydrogen-deuterium (H-D) exchange in the peptide groups combined with FT-IR spectroscopy.These experiments were conducted to investigate the origin of recently reported irreversible photobleaching in the temperature region below the two main thermal transition points of the purple membrane (PM).Reactivity measurements of bR with hydroxylamine in the dark revealed a high-temperature intermediate state defined by two transition points at ~60 and ~72 °C prior to the premelting of the PM.In the high-temperature intermediate state,remarkable enhanced water accessibility in the internal region of bR molecule was suggested despite no acceleration of H-D exchange in the main chain.A model for the high-temperature intermediate state of bR responsible for irreversible photobleaching was proposed.