Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, 00185, Rome, Italy.
Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123, Uppsala, Sweden.
Sci Rep. 2017 Oct 3;7(1):12593. doi: 10.1038/s41598-017-12827-0.
PDZ domains are one of the most important protein-protein interaction domains in human. While presenting a conserved three dimensional structure, a substantial number of PDZ domains display structural extensions suggested to be involved in their folding and binding mechanisms. The C-terminal α-helix extension (α3) of the third PDZ domain from PSD-95 (PDZ3) has been reported to have a role in function of the domain as well as in the stabilization of the native fold. Here we report an evaluation of the effect of the truncation of this additional helix on the folding and unfolding kinetics of PDZ3. Fluorescent variants of full length and truncated PDZ3 were produced and stopped-flow fluorescence measurements were made under different experimental conditions (pH, ionic strength and temperature) to investigate the folding kinetics of the respective variant. The results show that folding of PDZ3 is robust and that the mechanism is only marginally affected by the truncation, which contributes to a destabilization of the native state, but otherwise do not change the overall observed kinetics. Furthermore, the increase in the unfolding rate constants, but not the folding rate constant upon deletion of α3 suggests that the α-helical extension is largely unstructured in the folding transition state.
PDZ 结构域是人类最重要的蛋白质-蛋白质相互作用结构域之一。尽管具有保守的三维结构，但大量的 PDZ 结构域显示出结构延伸，这些延伸被认为参与了它们的折叠和结合机制。已经报道 PSD-95（PDZ3）的第三个 PDZ 结构域的 C 端α螺旋延伸（α3）在该结构域的功能以及天然折叠的稳定中具有作用。在这里，我们报告了评估该附加螺旋的截断对 PDZ3 折叠和展开动力学的影响。产生了全长和截断 PDZ3 的荧光变体，并在不同的实验条件（pH、离子强度和温度）下进行了停流荧光测量，以研究各自变体的折叠动力学。结果表明，PDZ3 的折叠是稳健的，并且该机制仅受到截断的轻微影响，这导致天然状态的不稳定，但不会改变整体观察到的动力学。此外，α3 缺失后解折叠速率常数的增加，但折叠速率常数没有增加，表明在折叠转变态中α-螺旋延伸大部分是无结构的。
