Folding and unfolding of a non-fluorescent mutant of green fluorescent protein.
Folding and unfolding of a non-fluorescent mutant of green fluorescent protein.
复制标题
绿色荧光蛋白非荧光突变体的折叠和展开。
DOI：
10.1088/0953-8984/19/28/285223
复制
发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Clark,PatriciaL
中科院分区：
文献类型：
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作者：
Kutrowska,BeataWielgus;Narczyk,Marta;Buszko,Anna;Bzowska,Agnieszka;Clark,PatriciaL
绿色荧光蛋白（GFP）来自太平洋水母a . victoria，由于其特定的发色团是在适当的蛋白质折叠后自发产生的，因此在生物技术和细胞分子生物学中作为蛋白质标记具有许多用途。在形成后，发色团非常稳定，在蛋白质展开过程中保持完整，这意味着GFP展开过程不是与原始折叠反应相反；也就是说，微观可逆性原理不适用。我们已经产生了突变体S65T/G67A-GFP，它不能有效地形成环状发色团，目的是研究GFP在完全可逆条件下的折叠、展开和竞争聚集。我们的研究是在盐酸胍（GdnHCl）存在下进行的。利用固有色氨酸荧光和1,1 '-双（4-苯胺-5-萘磺酸）（双- ans）荧光监测GFP构象。光散射用于跟踪GFP聚集。我们从这些荧光测量中得出结论，S65T/G67A-GFP折叠在很大程度上是可逆的。在平衡折叠过程中，第一步是形成一个易于聚集的熔融球。
Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; ie, the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride (GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1, 1'-bis (4-anilino-5-naphthalenesulphonic acid)(bis-ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation.