In-situ NMR characterization of the difference between the light and dark-adapted state of D94N HmbRI

• Main Authors: Yu-Ting Chang, 張宇廷
• Other Authors: Chun-Chung Chan
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/x522rh

碩士 === 國立臺灣大學 === 化學研究所 === 107 === A proton pump that builds up a proton gradient across a biological membrane is a membrane protein. A typical example is Haloarcula marismortui bacteriorhodopsin (HmbRI) that achieves its functionality as a proton pump by changing its conformation in the presence of green light. The study of the structural changes in membrane protein is crucial but difficult because of the problems such as sample preparation. In this thesis, we report that the purification of monomeric D94N HmbRI was improved by lowering the pH of the purification buffers and including an additional step of ionic-exchange chromatography, which can successfully and efficiently separate the monomeric and trimeric form of D94N HmbRI. Moreover, we found that the ratio between monomer and trimer can be manually controlled. With extensive detergent and buffer screening, we were able to obtain the 1H-15N TROSY HSQC spectrum of D94N HmbRI with good quality. By incorporating four optical fibers into a double layer NMR tube, the in-situ light coupling solution state NMR measurements were conducted. The lifetime of the light-adapted state and the M-state decay of D94N HmbRI was measured. The correlation between light intensity and the duration of light adaptation was further investigated. Using this device, the differences between the 1H-15N TROSY HSQC spectra for the dark-adapted state and the light-adapted state of D94N HmbRI were observed.