Dalessio Paula M, Fromholt Susan E, Ropson Ira J
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Proteins. 2005 Oct 1;61(1):176-83. doi: 10.1002/prot.20463.
Multiple phases have been observed during the folding and unfolding of intestinal fatty acid binding protein (WT-IFABP) by stopped-flow fluorescence. Site-directed mutagenesis has been used to examine the role of each of the two tryptophans of this protein in these processes. The unfolding and refolding kinetics of the mutant protein containing only tryptophan 82 (W6Y-IFABP) showed that the tryptophan at this location was critical to the fluorescence signal changes observed throughout the unfolding reaction and early in the refolding reaction. However, the kinetic patterns of the mutant protein containing only tryptophan 6 (W82Y-IFABP) indicated that the tryptophan at this location participated in the fluorescence signal changes observed early in the unfolding reaction and late in the refolding reaction. Together, these data suggest that native-like structure was formed first in the vicinity of tryptophan 82, near the center of the hydrophobic core of this beta-sheet protein, prior to formation of native-like structure in the periphery of the protein.
通过停流荧光法观察到肠脂肪酸结合蛋白（WT-IFABP）折叠和去折叠过程中的多个阶段。定点诱变已被用于研究该蛋白的两个色氨酸各自在这些过程中的作用。仅含有色氨酸82的突变蛋白（W6Y-IFABP）的去折叠和重折叠动力学表明，该位置的色氨酸对于整个去折叠反应以及重折叠反应早期观察到的荧光信号变化至关重要。然而，仅含有色氨酸6的突变蛋白（W82Y-IFABP）的动力学模式表明，该位置的色氨酸参与了去折叠反应早期和重折叠反应后期观察到的荧光信号变化。这些数据共同表明，在该β-折叠蛋白疏水核心中心附近的色氨酸82附近，首先形成了类似天然的结构，然后才在蛋白外围形成类似天然的结构。
