Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, Rome, Italy.
Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Naples, Italy.
J Biol Chem. 2023 Mar;299(3):102983. doi: 10.1016/j.jbc.2023.102983. Epub 2023 Feb 3.
Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multidomain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multidomain protein may either fold independently on each other or exhibit interdependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single cooperative unit. By exploiting quantitatively, the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short autoinhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multidomain systems.
尽管协同作用是折叠的一个既定且普遍的特性，但我们目前对多结构域折叠中这一特性的理解仍然相对有限。事实上，有相反的结果表明，多结构域蛋白的组成结构域可能彼此独立折叠，也可能表现出相互依存的超结构域现象。为了解决这个问题，我们在这里展示了对串联重复蛋白折叠的比较分析，该蛋白由两个连续的 PDZ 结构域组成，与它们各自孤立的结构域相比。通过详细分析不同实验条件下的平衡和动力学折叠，我们证明，尽管每个 PDZ 结构域在孤立状态下都能够独立折叠，但与以前表征的 PDZ 串联重复序列不同，全长构建体作为一个单一的协同单元折叠和解折叠。通过定量利用，将串联重复的折叠与观察到的组成结构域的折叠进行比较，以及通过表征缺乏短的自动抑制片段的截断变体，我们成功地合理化了观察到的协同作用的分子基础，并尝试为多结构域系统推断一些一般结论。
