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. Study holds ProTherm entries: 8431 Extra Details: CI2; foldon; folding intermediate; protein design
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:36 p.m.
|Number of data points||3|
|Proteins||Subtilisin-chymotrypsin inhibitor-2A ; Subtilisin-chymotrypsin inhibitor-2A|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence SSVEKKPEGVNTGAGDRHNLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG|