Takahashi Yusuke, Ebrey Thomas G
Department of Biology, University of Washington, Seattle 98195, USA.
Biochemistry. 2003 May 27;42(20):6025-34. doi: 10.1021/bi020629+.
Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.
此前我们报道了日本林蛙（Cynops pyrrhogaster）视网膜中脊椎动物视觉色素短波长敏感2（SWS2）家族成员的序列[高桥洋等人（2001年），《欧洲生物化学学会联合会快报》501卷，第151 - 155页]。现在我们已经表达了脱辅基色素并用A1视黄醛使其再生。其最大吸收波长为474 nm，与其他已知的SWS2色素（418 - 455 nm）相比有很大的红移。为了确定控制其光谱调谐的氨基酸残基，我们替换了靠近发色团且日本林蛙和牛蛙（最大吸收波长 = 430 nm）野生型SWS2色素之间存在差异的残基：Pro91Ser、Ser94Ala、Ile122Met、Cys127Ser、Ser211Cys、Tyr261Phe和Ala292Ser。这些定点突变体中的每一个都导致了日本林蛙色素的蓝移，其中五个引起了显著的位移；它们的总和大约等于牛蛙和日本林蛙色素最大吸收波长之间的差异，即44 nm。多个七残基突变体的最大吸收波长向442 nm的32 nm位移与野生型牛蛙色素相当接近。因此，我们替换的七个氨基酸残基是日本林蛙SWS2色素光谱红移的主要原因。其中两个残基，91和94，以前未被确定为视觉色素中的波长调节位点。其中一个，91，可能通过一种新机制调节颜色：改变通过水与发色团相连的氢键网络，在这种情况下是其抗衡离子Glu113。
