Studies Of The Mechanism Of Proton Transfer In Bacteriorhodopsin By Solid State Nuclear Magnetic Resonance

Bacteriorhodopsin(bR) is the sole integral membrane protein of the purple membrane in Halobacterium salinarum, functioning as the light-driven proton pump. The 26-kDa protein consists of 248 amino acids, which forms a single polypeptide containing a bundle of seven transmembrane spanning a-helixes, linked by short loops on either side of the cell membrane. bR encapsulates a retinal chromophore which is covalently linked to Lys-216 in helix G via a protonated Schiff base (SB). Light absorbed by the pigment drives the photoisomerization of the retinal chromophore from all trans to 13-cis, which lead to a vectorial transport of a proton across the membrane.Nuclear Magnetic Resonance (NMR) technique can only detect those nuclei that their spin number is not zero. And the spin of those major natural abundance nuclei, such as 13C,15N is zero. Therefore we need to label those nuclei to detect their NMR signal. In my experiment, we added some special isotope labeled amino acid in the synthetic media. Those amino acid can be absorbed by Halobacterium salinarum, and be integrated into the bacteriorhodopsin. We also optimized the culture conditions and the purification procedure of bacteriorhodopsin.We have made bacteriorhodopsin samples consist of 13C,15N labeled glycin and alanine and other non-labeled amino acid. We conduct 1-dimentional and 2-dimentional NMR experiments. We compared the CP and double quantum filtered experiment and find that the latter can filter the signals from those rare natural abundance nuclei with spin 1/2. This technique can be applied to bacteriorhodopsin samples with more amino acids that have been isotope labeled. And also, we conduct 2-dimentional RFDR experiment. Since the glycine have noβcarbon, which lead to the consequence that there is no cross peak between carbonyl carbon and (3 carbon. This can help us to specify the peak assignment.