The four-helix protein Im7 folds through an on-pathway intermediate at pH 7.0 and 10 degrees C. By contrast, under these conditions there is no evidence for a populated intermediate in the folding of its more stable homologue, Im9, even in the presence of 0.4 M sodium sulphate. Previous studies using phi-value analysis have shown that the Im7 intermediate is misfolded, in that three of its four native helices are formed, but are docked in a non-native manner. Using knowledge of the structure of the intermediate of Im7, we have used rational design to stabilise an intermediate formed during the folding of Im9 by the introduction of specific stabilising interactions at positions known to stabilise the Im7 folding intermediate through non-native interactions. We show that the redesigned Im9 sequence folds with three-state kinetics at pH 7.0 and have used phi-value analysis to demonstrate that this species resembles the misfolded intermediate populated during Im7 folding. The redesigned Im9 sequence folds 20-fold faster than the wild-type protein under conditions in which folding is two-state. The data show that intermediate formation is an important feature of folding, even for small proteins such as Im9 for which these partially folded states do not become significantly populated. In addition, they show that the introduction of stabilising interactions can lead to rapid refolding, even when the contacts introduced are non-native. Study holds ProTherm entries: 17235, 17236, 17237, 17238, 17239, 17240, 17241, 17242, 17243, 17244, 17245, 17246, 17247 Extra Details: histidine-tagged Im9,2 mM DTT and 1 mM EDTA were added in the experiment. immunity protein; folding; intermediate; rational design; non-native
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:49 p.m.
|Number of data points||24|
|Proteins||Colicin-E9 immunity protein ; Colicin-E9 immunity protein|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ionic:: , pH:7.0 ; Experimental Assay: dG_H2O pH:6.0, ionic:Na2SO4: 0.4 M ; Derived Quantity: ddG_H2O ionic:: , pH:7.0 ; Derived Quantity: ddG_H2O pH:6.0, ionic:Na2SO4: 0.4 M|
|Libraries||Mutations for sequence MELKHSISDYTEAEFLQLVTTICNADTSSEEELVKLVTHFEEMTEHPSGSDLIYYPKEGDDDSPSGIVNTVKQWRAANGKSGFKQG|