Rekas Agata, Alattia Jean-René, Nagai Takeharu, Miyawaki Atsushi, Ikura Mitsuhiko
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
J Biol Chem. 2002 Dec 27;277(52):50573-8. doi: 10.1074/jbc.M209524200. Epub 2002 Oct 4.
Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 A resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37 degrees C by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the Calpha-Cbeta bond of Tyr(66) during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site.
绿色荧光蛋白（GFP）的黄色发射变体因其发射光谱红移以及对pH和离子强度等环境参数敏感，已被发现在生物系统的各种应用中十分有用。然而，其成熟缓慢的特性以及对更强荧光的新要求使得对这些蛋白质进行进一步的诱变研究成为必要。Venus是一种新变体，具有更好的成熟度和亮度，以及更低的环境依赖性，它是最近通过在特征明确的变体增强型黄色荧光蛋白（EYFP）中引入五个突变而开发出来的。在本文中，我们展示了分辨率为2.2埃的Venus晶体结构，这使我们能够将其新特性与这些突变点联系起来。发现由F46L突变引发的发色团附近几个侧链的重排，通过消除可能阻碍多肽链折叠的空间位阻和能量限制，并通过在发色团形成过程中加速Tyr(66)的Cα-Cβ键的氧化，从而改善了37摄氏度时的成熟度。还发现M153T、V163A和S175G通过创造更大灵活性的区域来提高成熟速率。F64L在分子中诱导了大的构象变化，通过阻止离子进入结合位点从而消除了卤化物敏感性。
