Ervin John, Larios Edgar, Osváth Szabolcs, Schulten Klaus, Gruebele Martin
Department of Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Illinois 61801, USA.
Biophys J. 2002 Jul;83(1):473-83. doi: 10.1016/S0006-3495(02)75183-7.
Hyperfluorescent intensity maxima during protein unfolding titrations are often taken as a sign for a thermodynamic folding intermediate. Here we explore another possibility: that hyperfluorescence could be the signature of a "pretransition" conformationally loosened native state. To model such native states, we study mutants of a fluorescent ubiquitin variant, placing cavities at various distances from the tryptophan fluorophore. We examine the correlation between protein flexibility and enhanced fluorescence intensity by using circular dichroism, fluorescence intensity unfolding titrations, fluorescence anisotropy measurements, and molecular dynamics. Based on experiment and simulation, we propose a simple model for hyperfluorescence in terms of static and dynamic conformational properties of the native state during unfolding. Apomyoglobin denaturant unfolding and phosphoglycerate kinase cold denaturation are discussed as examples. Our results do not preclude the existence of thermodynamic intermediates but do raise caution that by itself, hyperfluorescence during unfolding titrations is not conclusive proof of thermodynamic folding intermediates.
蛋白质解折叠滴定过程中荧光强度最大值通常被视为热力学折叠中间体的标志。在此，我们探讨另一种可能性：超荧光可能是“预转变”构象松弛天然态的特征。为了模拟此类天然态，我们研究了一种荧光泛素变体的突变体，在距色氨酸荧光团不同距离处设置空穴。我们通过圆二色性、荧光强度解折叠滴定、荧光各向异性测量和分子动力学来研究蛋白质柔韧性与增强荧光强度之间的相关性。基于实验和模拟，我们根据解折叠过程中天然态的静态和动态构象性质，提出了一个关于超荧光的简单模型。以脱辅基肌红蛋白变性剂解折叠和磷酸甘油酸激酶冷变性为例进行了讨论。我们的结果并不排除热力学中间体的存在，但确实提醒人们，仅靠解折叠滴定过程中的超荧光本身并不能确凿证明热力学折叠中间体的存在。
