不同吸收波長蛋白在大腸桿菌的表現

Abstract

GFP-like-protein family由11 β-sheet 所構成的β-can結構，並以一α-helix貫穿，β-can中央α-helix上的residue可形成chromophore結構，為此類蛋白能發出螢光或具有色澤的蛋白質．但多數研究著重於改善此類蛋白質性質，或改變為不同色澤的螢光蛋白，鮮少探討non-fluroescence chromoprotein的性質及其色澤與胺基酸之間的關係．此研究利用色澤蛋白研究胺基酸的突變對於蛋白質色澤的影響．於蛋白質多處進行單點突變，發現將蛋白質chromphore 上第一個胺基酸突變為S、L時，蛋白質由紫紅色變為粉紅色及淺膚色，吸收光譜則有藍移10-60 nm的情形，且突變為L的除色澤上的改變外，在受短波長的光照射會放出青藍色螢光；除chromophore序列外，對胺基酸39的位置由Q改為S時也造成蛋白質改變為橘紅色，吸收光譜藍移60 nm；進行雙點突變時發現突變蛋白質Y64L/I196H為黃色，且在受短波長的光激發後放出螢光．另外利用Random mutation的方式，分別取得I70V及T194I兩藍紫色突變蛋白質，其吸收光譜則有紅移的現象．可知色澤蛋白受胺基酸的所影響．

The GFP-like protein family is the proteins that are consisted of an 11-stranded β-can cylinder with a central axis of α-helix. The chromophore is located at the α-helix, which is the main structure to exhibit either fluorescences or colors. Most previous researches were focused on trying to improve the biological properties of the GFP-like proteins such as aggregation, oligomerization, photostability and fluorescent color palette. However, little is known which position and property of amino acid of protein might affect the color appearance. In this study, we attempted to perform mutagenesis to know whether the mutated amino acids can manipulate the color image. We found when the first amino acid of chromophore was replaced by either S or L, the color of protein was changed into to pink or skin color and the maximum absorption peak was blue-shifted. Furthermore, when the amino acid at 39 was replaced by S, the color of protein was changed into orange and the maximum absorption peak was red-shifted. Moreover, using random mutagenesis strategy, we found that amino acids at 70 and 194 were mutated to V and I, respectively, the color of protein was changed into purple blue. Based on the line of evidences, we concluded that the color exhibition of chromoprotein was completely dependent on both the position and the property of amino acid residues located at chromoprotein.