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.
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.
J Mol Biol. 2021 Apr 2;433(7):166862. doi: 10.1016/j.jmb.2021.166862. Epub 2021 Feb 1.
Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.
尽管人类蛋白质组的绝大多数都由多结构域蛋白组成，但多结构域折叠和错误折叠的研究仍然是一个相对未被充分探索的领域。Whirlin 蛋白是一种在内耳中表达的多结构域支架蛋白。它的特点是 PDZ 结构域串联重复。Whirlin 的前两个 PDZ 结构域（PDZ1 和 PDZ2，即 P1P2）结构上很接近，由一段无序的短连接子隔开。我们最近描述了 P1P2 串联的折叠机制。两个结构域热力学稳定性的差异使我们能够选择性地展开一个或两个 PDZ 结构域，并确定错误折叠中间产物的积累，我们证明该中间产物保留了生理结合活性。在这项工作中，我们对 P1P2 的折叠和展开进行了广泛的表征。基于观察到的数据，我们描述了一个综合的动力学分析，该分析令人满意地拟合了实验，并提供了一个有价值的模型来解释多结构域折叠。本研究中描述的实验和分析方法可能对解释复杂的多结构域蛋白折叠动力学具有普遍意义。
