서강대학교

초록/요약

Many microbes contain retinal proteins which is a seven-transmembrane helices protein, called rhodopsin. It shows several functions such as ion pump, channel, and sensory receptor using light. Donghaeana (Nonlabens) dokdonensis DSW-6, classified into a marine flavobacterium and collected from the surface seawater near Dokdo island, had two types of rhodopsins that were supposed to pump either sodium ion and proton. For the sodium-pumping rhodopsin, it was named Donghaeana dokdonensis rhodopsin 2 (DDR2) that had NDQ motif and absorbs λmax = 528 nm at pH 7.0. In this study, I tried to understand spectroscopic and functional characteristics of DDR2 in detail. Using an error-prone PCR and mutant screening methods, I found that DDR2 D116G mutant absorbed λmax = 580 nm at pH 7.0 but lost pumping activity. In order to make DDR2 D116G to get pumping activity, I introduced several negative amino acid residue in the protein. I found interesting mutant that had more 65 nm bathochromic shifted λmax than wild-type. I characterized the DDR2 N112E / D116G / Q123E mutant by using protein 3D structure modeling, site-directed mutagenesis, and pumping analysis which absorbed λmax = 593 nm at pH 7.0 and transports lithium ion in 100 mM LiCl. All methods and ideas has significance that the selection of red-shifted λmax mutants and reconstitution of pumping mechanisms are also applicable to any other rhodopsins.