Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
J Biol Chem. 2010 Feb 12;285(7):4859-69. doi: 10.1074/jbc.M109.000695. Epub 2009 Nov 9.
Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of (15)N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the beta-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.
Venus 是一种黄色荧光蛋白，因其快速的发色团成熟率和明亮的黄色荧光而被开发出来，这种荧光对 pH 值和离子浓度的变化相对不敏感。在这里，我们使用化学变性剂和各种光谱探针，在 pH 值为 6.0 到 8.0 的范围内，对 Venus 的稳定性和折叠进行了详细研究。通过在 13 个月内使用 NMR 光谱法跟踪 (15)N 标记 Venus 的氢氘交换，确定了一些高度保护酰胺基团的残基特异性展开自由能。一些酰胺基团的交换速率低于每年一次。确定了 Venus 的超稳定核心，并与先前报道的绿色荧光蛋白进行了比较。这些结果是根据荧光蛋白的稳定性和折叠来讨论的。在酸性条件下，我们发现 Venus 在相对较低浓度的盐酸胍下，黄色荧光会急剧下降。对这种效应的详细研究表明，这是由于离子与蛋白质之间的 pH 依赖性非特异性相互作用。与先前对增强型绿色荧光蛋白变体 S65T/T203Y 的研究不同，该变体显示出特定的卤素离子结合位点，NMR 化学位移映射显示没有特定离子结合的证据。相反，观察到许多残基的化学位移扰动，这些残基主要位于β桶结构的两个盖子中，这表明在酸性条件下增加离子强度时会发生小范围的结构重排，并且这些结构重排会传播到发色团，导致荧光猝灭。
