Chirico G, Cannone F, Diaspro A
Department of Physics, University of Milano Bicocca, Piazza della Scienza 3, Milano, Italy.
Eur Biophys J. 2006 Oct;35(8):663-74. doi: 10.1007/s00249-006-0075-5. Epub 2006 Jun 20.
We have studied the unfolding of single molecules of GFP-mut2 mutant trapped in wet silica gels in a wide range of GuHCl concentration. After the addition of denaturant, the number of fluorescent molecules decreases with unfolding rates (of the order of 0.01 min(-1)) that are in very good agreement with bulk fluorescence and circular dichroism data. Unexpectedly, single molecule experiments show rare fluctuations in the number of fluorescent proteins at equilibrium. On the other hand, although a first approximate description of the number decays can be reasonably performed by single exponential functions, the distributions of the single molecule unfolding times show a maximum at times congruent with 50-100 min up to the denaturation midpoint concentration of [GuHCl] congruent with 2.5 M. A theoretical analysis of the distributions indicates that this feature is a fingerprint of the competition between unfolding and refolding processes when the protein is very far from the midpoint denaturant concentration.
我们研究了被困在湿硅胶中的绿色荧光蛋白突变体GFP-mut2单分子在广泛的盐酸胍（GuHCl）浓度范围内的解折叠情况。加入变性剂后，荧光分子的数量随着解折叠速率（约为0.01 min⁻¹）而减少，这与体相荧光和圆二色性数据非常吻合。出乎意料的是，单分子实验表明，在平衡状态下荧光蛋白的数量存在罕见的波动。另一方面，虽然单指数函数可以对数量衰减进行合理的初步近似描述，但单分子解折叠时间的分布在与50 - 100分钟相当的时间处出现最大值，直至变性中点浓度[GuHCl]约为2.5 M。对这些分布的理论分析表明，当蛋白质距离变性剂中点浓度很远时，这一特征是解折叠和重折叠过程之间竞争的标志。
