Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: A steady-state and time-resolved study of yeast phosphoglycerate kinase
Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: A steady-state and time-resolved study of yeast phosphoglycerate kinase
复制标题
DOI：
10.1021/bi9707887
复制
发表时间：
1997-09-16
期刊：
影响因子：
2.9
通讯作者：
Mas, MT
中科院分区：
文献类型：
--
作者：
Lillo, MP;Beechem, JM;Mas, MT
为研究蛋白质折叠反应，建立了基于多点距离的荧光共振能量转移分析系统。利用单半胱氨酸和双半胱氨酸取代突变的方法，在酵母磷酸甘油酸激酶(PGK)的三级结构中放置了巯基残基。这些反应性半胱氨酸被外源供体[5-[[2-(2-碘乙酰氨基)乙基]氨基]-1-萘磺酸]和受体(5-碘乙酰氨基荧光素)共价修饰，并遵循了详细的实验策略，结果表明，当这些相对较大的外源荧光标记共价连接到适当选择的溶剂暴露残基上时，它们不影响蛋白质的内在稳定性。将PGK晶体结构与蛋白质和时间分辨各向异性实验中内置的染料的分子动力学模拟相结合，以估计每个供体/受体对的更现实的取向因子[kappa(2)](*)。时间分辨和稳态荧光能量转移实验表明，这种跨越六个不同供体-受体距离的距离分析在30-70埃的范围内是线性的和准确的(在10-20%以内)。这种PGK的距离分析系统允许测量蛋白质折叠反应过程中域内和域间空间组织的长期变化。我们开发的方法可以应用于任何蛋白质系统，在该系统中，唯一的一个和两个半胱氨酸残基可以被工程化为蛋白质。在接下来的文章中[Lillo，M.P.，et al.(1997年)生物化学36,11273-11281]，这些多位能量转移对被用于停流展开研究。
A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions. Single-and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK). These reactive cysteines were covalently modified with extrinsic donor [5-[[2-(2-iodoacetamido)ethyl]amino]-1- naphthalenesulfonic acid] and acceptor (5-iodoacetamidofluorescein) fluorescent labels, A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein. The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, [kappa(2)](*), for each donor/acceptor pair. Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 Angstrom. This distance assay system for PGK allows for the measurement of long-range changes in intra-and interdomain spatial organization during protein folding reactions. The approach which we have developed can be applied to any protein system in which unique one-and two-site cysteine residues can be engineered into a protein. In the following paper [Lillo, M. P., et al. (1997) Biochemistry 36, 11273-11281], these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.