Inaba K, Kobayashi N, Fersht A R
Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge, CB2 2QH, UK.
J Mol Biol. 2000 Sep 8;302(1):219-33. doi: 10.1006/jmbi.2000.4024.
Chymotrypsin inhibitor 2 (CI2) is the archetypal single-foldon protein that folds in simple two-state kinetics without the accumulation of a folding intermediate. To model the effects of fusion of single foldons to give a multi-foldon protein, we engineered a "double-CI2" protein, in which another CI2 polypeptide was inserted into the loop region of the parent CI2. CD and HSQC spectra demonstrated that while the double-CI2 protein adopted two kinds of native conformations, CI2-like structure was almost preserved in both the domains of double-CI2. In the folding kinetic studies, double-CI2 exhibited a remarkable rollover of the observed folding rates at low denaturant concentrations, indicating that double-CI2 accumulated a kinetic folding intermediate. The different folding mechanisms between WT-CI2 and double-CI2 support the present view that protein size or number of domains is an important determinant for formation of folding intermediates.
糜蛋白酶抑制剂2（CI2）是典型的单折叠子蛋白，以简单的两态动力学折叠，不积累折叠中间体。为了模拟单折叠子融合产生多折叠子蛋白的效果，我们设计了一种“双CI2”蛋白，其中另一个CI2多肽被插入到亲本CI2的环区域。圆二色光谱（CD）和异核单量子相干谱（HSQC）表明，虽然双CI2蛋白采用两种天然构象，但在双CI2的两个结构域中，CI2样结构几乎都得以保留。在折叠动力学研究中，双CI2在低变性剂浓度下表现出明显的观察到的折叠速率翻转，表明双CI2积累了一个动力学折叠中间体。野生型CI2（WT-CI2）和双CI2之间不同的折叠机制支持了目前的观点，即蛋白质大小或结构域数量是折叠中间体形成的重要决定因素。
