Photoirradiation and Microwave Irradiation NMR Spectroscopy

Abstract

In situ photoirradiation solid-state nuclear magnetic resonance (NMR) spectroscopy is designed for optical irradiation from the top part of a zirconia rotor through a glass cap, which makes it possible to efficiently irradiate the inside of the rotor. This experimental method has made it possible to observe photo-intermediates of sensory rhodopsins, such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII), and bacteriorhodopsin (bR) Y185F mutant. In SRI, green light generates M-intermediates, which exhibit positive phototaxis, while blue light generates P-intermediates, which exhibit negative phototaxis. In SRII, green light generates M-intermediates and blue light generates O-intermediates. In Y185F-bR, O-intermediates were first observed using solid-state NMR spectroscopy. The microwave irradiation NMR spectrometer was developed in-house by modification of a commercial NMR spectrometer. A flat long copper ribbon was used as a capacitor and a half turn of copper ribbon at the edge was used as an inductor for the microwave resonance circuit, which was coaxially inserted inside the radiofrequency induction coil and allowed NMR signals to be observed under microwave irradiation conditions. The temperature of N-(4-methoxybenzylidene)-4-butylaniline (MBBA) during microwave irradiation was estimated by measuring the temperature-dependent chemical shifts, whereby different protons were found to indicate significantly different temperatures in the molecule. Liquid crystalline-isotropic phase correlation 2D NMR spectra were observed using pulsed microwave irradiation for rapid temperature jump experiments.