Barondeau David P, Tainer John A, Getzoff Elizabeth D
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
J Am Chem Soc. 2006 Mar 15;128(10):3166-8. doi: 10.1021/ja0552693.
The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification.
维多利亚多管水母绿色荧光蛋白（GFP）通过一种非凡的翻译后修饰，由其组成氨基酸Ser65、Tyr66和Gly67形成一个荧光团，该修饰涉及自发的肽主链环化、脱水和氧化反应。在此，我们通过将化学还原和厌氧方法与动力学分析及蛋白质结构测定相结合，来测试并拓展对荧光团生物合成的理解。经连二亚硫酸盐处理的GFP变体的两个高分辨率结构揭示了一种以前未被表征的发色团烯醇盐中间体形式，该中间体在暴露于空气时能够生成荧光团（半衰期 = 39分钟-1）。现在，分离这种烯醇盐中间体将允许对GFP及其红色荧光蛋白同源物中荧光团生物合成的限速氧化步骤进行特异性探究。这种有针对性的表征可能会促成设计出成熟更快的蛋白质，在生物技术和细胞生物学中有更多应用。此外，我们的结果揭示了GFP蛋白环境如何通过稳定原本高能的烯醇盐中间体来实现其翻译后修饰，从而模拟酶系统。
