Design Of Fluorescent Probe Based On The Lov2 Domain And Study Of Biliverdin Interaction With The Dna

The phototropin LOV（Light, Oxygen, Voltage） domain from the oat contains two subunits, LOV1 and LOV2. While LOV2 domain acts as a kind of fluorescent protein, comparing with GFP（green fluorescent protein）, possesses its unique advantages: 1. LOV2 domain is soluble; 2. Its relative molecular mass is small, about only 100-140 amino acid residue; 3. Its light absorption cofactor-FMN（flavin mononucleotide） almost exists in all varieties of cell; 4. Under the same condition, its fluorescence intensity is remarkably stronger than GFP’s. It is the merits that makes LOV2 a ideal tool in optogenetics study. Hence, it widely applied to the medical science and bioscience study nowadays.In this paper, it mainly includes three parts:1. With the idea from the oat phototropin gene sequence（Genbank）, LOV2 domain encoded gene fragment, AsLOV2（404-560）, was synthesized in this project; the cysteine 450 residue was then mutated to alanine in AsLOV2（404-560, C450A）. Then AsLOV2（404-521, C450A） was further produced by cutting Jα peptides on C–terminal. All of three gene fragments were cloned into the expression vector pGEX-6p-1, expressed by E.coli BL21（DE3）. After separation by glutathione sepharose 4B gel, gst fusion proteins were collected. The three purified proteins had similar absorption spectrum with that in the literature, indicating their photoactivities by combining with FMN in bacterial cells. The three proteins exhibited huge difference in the fluorescence properties, AsLOV2（404-560, C450A） showed 6 times stronger fluorescence than that of wild type. AsLOV2（404-521, C450A） showed 7 times stronger fluorescence than that of wild type. Based on that, the fusion protein of AsLOV2（404-521, C450A） and cytb562（cytochrome b562） was also constructed, namely, cytb562-3c-AsLOV2（404-521, C450A） and AsLOV2（404-521, C450A）-3c-cytb562. 3c protease（PreScission protease） has the effect of specifically identifying linker peptides in the fusion protein. The fusion protein was able to be cleaved by 3c protease. It turned out that the fusion protein was already cleaved by 3c protease, showing through the SDS-polyacrylamide gel electrophoresis figure, but the fluorescence intensity of fusion protein was not remarkably enhanced as was expected.2. In this study, more amino acids around cysteine 450 of AsLOV2 domain were mutated to cysteine, namely, constructing N449C、R451C、Q446C、I428C、F429C、A430C mutants, and so on. In the presence of Hg²⁺, since cysteine 450 thiol residue of wild type AsLOV2 could strongly coordinate with Hg²⁺ to enhance fluorescence, insteading of reacting with FMN, a special probe of Hg²⁺ could be achieved. However, these AsLOV2 mutants and wild type fluorescence intensity both did not be enhanced via Hg²⁺. However, F429 C mutant fluorescence intensity was slightly enhanced when Hg²⁺ was added.3. It also studied the interactions between BV（biliverdin） and DNA with different structure. It was indicated that BV could selectively bind to parallel G-quadruplex with increased UV absorption and induced a new peak at 740 nm. The 1:1 binding mode was confirmed by Job plot method. However, the complex showed weak fluorescence and couldn’t be detected.