Laboratory of Structural dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave, St. Petersburg 194064, Russia.
Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya str., 29, St. Petersburg 194064, Russia.
Int J Mol Sci. 2018 Sep 15;19(9):2776. doi: 10.3390/ijms19092776.
Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome BphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter , was found to have a different residual structure in the denaturants used.
近红外荧光蛋白（NIR FPs）基于细菌光感受器与天然胆绿素发色团的复合物，被广泛用作遗传编码的光学探针，用于可视化细胞过程和活体动物细胞和器官的深层组织成像。在这项工作中，我们表明，iRFP713 的各种光谱特性的稳态和动力学依赖性在蛋白质展开诱导下有很大不同，该蛋白质展开由盐酸胍（GdnHCl）、硫氰酸胍（GTC）和尿素引起。对 iRFP713 的三种单色氨酸突变体形式的展开研究表明，蛋白质的展开始于天然二聚体的解离，而单体仍然保持紧凑。进一步增加变性剂浓度会导致 iRFP713 形成具有暴露在蛋白质表面的疏水区的中间状态（I）。随着 GdnHCl 和 GTC 浓度的增加，iRFP713 的总表面电荷（pI 5.86）从负变为正，这是因为谷氨酸和天冬氨酸的负电荷通过羧基与 GdnH⁺离子之间形成盐桥而被中和，并且胍离子与谷氨酰胺和天冬酰胺的酰胺基团结合。积累 iRFP713 的中间状态的变性剂浓度与该状态下蛋白质表面电荷中和的 GdnH⁺离子浓度相吻合，导致强烈的蛋白质聚集。这显然是通过 iRFP713 用 GTC 展开来实现的。在 GdnHCl 展开蛋白质时，在较高的变性剂浓度下填充中间状态，并且不会观察到强烈的聚集。正如预期的那样，在存在尿素的情况下不会形成蛋白质聚集体。大分子表面电荷中和时蛋白质的聚集是蛋白质中间状态的主要指标。iRFP713 的展开状态，其形成伴随着参数 的显著下降，在使用的变性剂中发现具有不同的残留结构。
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