Andrews Benjamin T, Schoenfish Andrea R, Roy Melinda, Waldo Geoffrey, Jennings Patricia A
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093-0375, USA.
J Mol Biol. 2007 Oct 19;373(2):476-90. doi: 10.1016/j.jmb.2007.07.071. Epub 2007 Aug 15.
Many green fluorescent protein (GFP) variants have been developed for use as fluorescent tags, and recently a superfolder GFP (sfGFP) has been developed as a robust folding reporter. This new variant shows increased stability and improved folding kinetics, as well as 100% recovery of native protein after denaturation. Here, we characterize sfGFP, and find that this variant exhibits hysteresis as unfolding and refolding equilibrium titration curves are non-coincident even after equilibration for more than eight half-lives as estimated from kinetic unfolding and refolding studies. This hysteresis is attributed to trapping in a native-like intermediate state. Mutational studies directed towards inhibiting chromophore formation indicate that the novel backbone cyclization is responsible for the hysteresis observed in equilibrium titrations of sfGFP. Slow equilibration and the presence of intermediates imply a rough landscape. However, de novo folding in the absence of the chromophore is dominated by a smoother energy landscape than that sampled during unfolding and refolding of the post-translationally modified polypeptide.
许多绿色荧光蛋白（GFP）变体已被开发用作荧光标签，最近还开发了一种超级折叠GFP（sfGFP）作为一种强大的折叠报告蛋白。这种新变体显示出更高的稳定性和改进的折叠动力学，并且变性后天然蛋白的回收率达到100%。在这里，我们对sfGFP进行了表征，发现即使根据动力学解折叠和重折叠研究估计经过超过八个半衰期的平衡后，该变体在解折叠和重折叠平衡滴定曲线中仍表现出滞后现象，因为两者并不重合。这种滞后现象归因于被困在一种类似天然的中间状态。针对抑制发色团形成的突变研究表明，新的主链环化作用是sfGFP平衡滴定中观察到的滞后现象的原因。缓慢的平衡和中间体的存在意味着存在一个粗糙的能量景观。然而，在没有发色团的情况下从头折叠主要由比翻译后修饰多肽解折叠和重折叠过程中所采样的更平滑的能量景观主导。
