Dipartimento di Fisica, Università degli Studi di Milano-Bicocca, Italy.
Biophys Chem. 2011 Aug;157(1-3):24-32. doi: 10.1016/j.bpc.2011.04.004. Epub 2011 Apr 13.
We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.
我们使用荧光相关光谱和荧光各向异性衰减等荧光光谱技术，在纳秒到秒的宽时间范围内，研究了胍盐酸盐诱导的 GFPMut2 及其点突变 H148G 的解折叠动力学，这对于 GFP 的光化学和光物理是相关的。该突变影响 GFP 的解折叠动力学，导致在碱性 pH 值下更快的过程，其中质子动力学可以忽略不计，这可以归因于 His148 的双重作用，既作为向发色团的质子快门，又作为构象稳定剂。对于这两种突变体，我们发现一个位于β-链 3 附近的柔软区域，在变性剂浓度远低于使发色团荧光熄灭所需的浓度时，在 ns 范围内开始获得柔韧性，这可以从与蛋白质共价结合的外源探针的各向异性衰减中得出。
