Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy
Highly Heterogeneous Nature of the Native and Unfolded States of the B Domain of Protein A Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy
复制标题
二维荧光寿命相关光谱揭示 A 蛋白 B 结构域的天然和未折叠状态的高度异质性
DOI：
10.1021/acs.jpcb.7b00546
复制
发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Tahara Tahei
中科院分区：
文献类型：
--
作者：
Otosu Takuhiro;Ishii Kunihiko;Oikawa Hiroyuki;Arai Munehito;Takahashi Satoshi;Tahara Tahei
阐明蛋白质折叠机制对于理解蛋白质如何获得其独特的结构以实现各种生物学功能至关重要。为此，人们对小球状蛋白的折叠/展开进行了广泛的研究。有趣的是，最近的研究表明，即使是这样小的蛋白质也代表了相当复杂的过程。在本研究中，我们使用10 μs时间分辨率的二维荧光寿命相关光谱研究了小α-螺旋蛋白a的B结构域（BdpA）在平衡状态下的折叠/展开过程。结果表明，虽然BdpA是一个双态文件夹，但其原生态和未折叠态都是高度异构的，并且每个包系的构象转换发生在10 μs以内。结果表明，随着变性剂浓度的增加，两种体系的平均结构逐渐发生变化，并逐渐拉长。对两个突变体的分析表明，n端螺旋的磨损是原生态不均匀性的根源。由于在单分子水平上直接观察到天然态的系综性质尚未报道，因此本研究获得的数据为小分子蛋白质的复杂构象性质提供了新的见解。
Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small proteins represent considerably complex processes. In this study, we examined the folding/unfolding process of a small α-helical protein, the B domain of protein A (BdpA), at equilibrium using two-dimensional fluorescence lifetime correlation spectroscopy with 10 μs time resolution. The results showed that although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 μs. Furthermore, it was shown that the average structures of both ensembles gradually change and become more elongated as the denaturant concentration increases. The analysis on two mutants suggested that fraying of the N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single-molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins.