Detection and resolution of intermediate species in protein folding processes using fluorescence and circular dichroism spectroscopies and multivariate curve resolution
Detection and resolution of intermediate species in protein folding processes using fluorescence and circular dichroism spectroscopies and multivariate curve resolution
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DOI：
10.1021/ac025914d
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发表时间：
2002-12-01
影响因子：
7.4
通讯作者：
Tauler, R
中科院分区：
文献类型：
--
作者：
Navea, S;de Juan, A;Tauler, R
已经对α-乳白蛋白和α-脱乳清蛋白研究了热诱导的蛋白质展开/折叠过程。通过荧光和圆二色光谱技术对α-脱乳清蛋白进行监测的实验表明形成了中间物质，而在α-乳清蛋白的情况下，未检测到该中间物质。该中间物种的存在和分辨率，其光谱，以及蛋白质展开/折叠过程中所有构象的演变估计使用多元曲线分辨率交替最小二乘法。还阐明了每个解析蛋白质构象（包括中间体）中不同二级结构基序的性质和贡献。多变量分辨率已被证明是一个很好的工具，用于完整表征所有蛋白质构象参与折叠过程，包括中间物种，不能通过物理或化学手段分离。事实上，正是在这些中间体的测定和建模中，这种化学计量学方法在功率和可靠性方面优于基于更简单的测量和数据处理的先前方法，并填补了与蛋白质折叠过程中复杂机制的阐明和解释相关的空白。
Thermally induced protein unfolding/folding processes have been studied on alpha-lactalbumin and alpha-apolactalbumin. Experiments monitored by fluorescence and circular dichroism spectroscopic techniques on alpha-apolactalbumin showed the formation of an intermediate species, whereas in the case of a-lactalbumin, this intermediate species was not detected. The presence and resolution of this intermediate species, its spectrum, and the evolution of all conformations during protein unfolding/folding processes were estimated using the multivariate curve resolution-alternating least-squares method. Elucidation of the nature and contribution of the different secondary structure motifs in each of the resolved protein conformations, including the intermediate, was also carried out. Multivariate resolution has shown to be an excellent tool for the complete characterization of all protein conformations involved in folding processes, including intermediate species that cannot be isolated by physical or chemical means. Indeed, it is in the determination and modeling of these intermediates that this chemometric approach outperforms in power and reliability previous methodologies based on simpler measurements and data treatments and fills the void linked to the elucidation and interpretation of complex mechanisms in protein folding processes.