Single Residue Mutations at the EF Loop Affect the Properties of the Photocurrent and Dynamic Absorbance Spectrum of Halobacterium salinarum Bacteriorhodopsin

• Main Authors: Ming-Jin Jheng, 鄭明晉
• Other Authors: 楊啟伸
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/48959827458976026008

碩士 === 國立臺灣大學 === 生化科技學系 === 101 === Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) of Halobacteriun salinarum. BR is composed of seven transmembrane α-helices and contains one all-trans retinal chromophore which is covalently bound to the ϵ-amino group of the Lys 216 residue via a protonated Schiff base (PSB). Illumination of BR generates an electrochemical proton gradient across the PM by vectorial translocation of a proton from the cytoplasmic (CP) side to the extracellular (EC) side. Upon light absorption, the all-trans retinal isomerizes around the C13=C14 double-bond and reverts thermally back to the initial all-trans state, passing a series of intermediates named K, L, M, N, and O. The photocycle involves a proton transport process. During the L to M intermediate transition, a proton is released to the extracellular space from the proton releasing complex (PRC) which is composed of Arg 82, Glu 194, Glu 204, and internal H2O. During the N to O intermediate transition, the deprotonated Asp 96 uptake a proton from the cytoplasmic space. The transient pH change due to the light-induced proton release and uptake is usually studied by using a pH-sensitive dye whose absorption depends on pH. However, the restriction of the measurement is that the medium pH should be close to the pKa of the dye. Hence, a device is needed for the detection of the light-induced transient pH change in a wide pH range. When illumination with a continuous wave (CW) laser pulse, a photocurrent is detected from the BR-based photoelectrochemical cell. At neutral pH, the positive photocurrent is observed when the CW light source is turned on, and the negative photocurrent is observed when the CW light source is turned off. The photocurrent is investigated for the transient proton concentration changes generated by BR. In previous study, it is found that the rates of the proton uptake decrease in the E161C and R164C mutant proteins using the pH-sensitive dye pyranine. In the first part of my work, I compare the photoresponse of the E161C and R164C mutations with the wild type BR. It is found that the decay rates of the light-on phase as well as the recovery rates of the light-off phase increase in the E161C and R164C mutations. These results suggest that the origin of the decay of the light-on phase as well as the recovery of the light-off phase are associated with the proton uptake of BR, and thus can be used for the investigation of the proton uptake rates of the wild type and mutant proteins. According to structural dynamic studies, significant conformational changes of the EF loop are observed during the photocycle of BR which means EF loop is likely critical in the function of BR. In the second part, I construct mutantions containing cysteine substitutions located in the EF loop of BR. Each of 10 residues, located in the EF loop of BR, was individually replaced by cystein, respectively. It is found that the rates of the proton uptake increase in the mutantions near the E-helix and F-helix. It means that EF loop play an important role during the proton uptake of BR.