Seifert Markus H J, Ksiazek Dorota, Azim M Kamran, Smialowski Pawel, Budisa Nediljko, Holak Tad A
Max-Planck-Institute for Biochemistry, 82152 Martinsried, Munich, Germany.
J Am Chem Soc. 2002 Jul 10;124(27):7932-42. doi: 10.1021/ja0257725.
Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.
绿色荧光蛋白及其突变体已成为分子生物学中有价值的工具。它们还提供了富含光物理和光化学现象的体系，了解这些现象对于绿色荧光蛋白新的优化变体的开发很重要。令人惊讶的是，迄今为止尚未有关于绿色荧光蛋白的核磁共振研究报道，这可能是因为它们极易聚集。在此，我们报告了对用氟化色氨酸标记的绿色荧光蛋白（GFP）和青色荧光蛋白（CFP）突变体进行的¹⁹F核磁共振（NMR）研究，该研究能够检测这些蛋白质中的慢分子运动。动态核磁共振和¹⁹F弛豫测量的协同使用，辅以温度、浓度和折叠依赖性实验，为青色荧光蛋白两种不同构象状态之间存在缓慢交换过程提供了直接证据。¹⁹F NMR弛豫和线形分析表明，这些状态之间的交换时间尺度在1.2 - 1.4毫秒范围内。热力学分析显示，在T = 303 K时，参与交换过程的两种状态的焓变ΔH₀ = (18.2 ± 3.8) kJ/mol，熵变TΔS₀ = (19.6 ± 1.2) kJ/mol，表明存在熵焓补偿。活化自由能估计约为60 kJ/mol。发色团本身或更可能是与之密切相关的组氨酸148的两种构象之间的交换，被认为是绿色荧光蛋白构象流动性的结构基础。本研究中产生一系列单原子交换（“原子突变”）如H → F的可能性，为通过核磁共振表征和量化蛋白质中的动态过程提供了一种有用的方法。
