Molecular Cloning, Structural Characterization and Color-regulating Mechanism of Novel Chromoprotein Isolated from Marine Organisms
Date Issued
2015
Date
2015
Author(s)
Chiang, Cheng-Yi
Abstract
GFP-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated novel GFP-like chromoproteins from the carpet anemone Stichodactyla haddoni and Stichodactyla gigantea, termed shCP and sgBP, respectively. The shCP protein has maximum absorption wavelength peak (λmax) is located at 574 nm, resulting in a purple appearance. The sgBP has a λmax at 608 nm and shows a blue appearance. We mutated aa residues to enhance the color properties according to the simulated structure of shCP and the crystal structure of sgBP. With the mutations in and surrounding 5 Å of the chromophore, we found that the mutated proteins exhibited altered optical properties. The λmax of chromophore-mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. shCP with surrounding mutations Q39S and T194I also exhibited coloration changes to red and purple-blue. An additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In the chromoprotein sgBP, the surrounding S157C mutation shifted the λmax to 604 nm and darkened the blue color expression, indicating the color expression of chromoprotein could be altered by deprotonation state change of the phenolic group in the chromophore. Additionally, we found that the cDNAs of shCP, sgBP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos. Our results provide a structural basis for the color regulation of the biomarker development.
Subjects
chomoprotein
biomarker
protein engineering, transgenesis
Type
thesis
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