Switching two-state to three-state kinetics in the helical protein Im9 via the optimisation of stabilising non-native interactions by design.


Abstract

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

Submission Details

ID: 4quuDDMP

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Friel CT;Beddard GS;Radford SE,J. Mol. Biol. (2004) Switching two-state to three-state kinetics in the helical protein Im9 via the optimisation of stabilising non-native interactions by design. PMID:15313622
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1IMP 1997-09-17 COLICIN E9 IMMUNITY PROTEIN IM9, NMR, 21 STRUCTURES
2K5X 2008-12-09 Chemical shift structure of COLICIN E9 DNASE domain with its cognate immunity protein IM9
1E0H 2000-10-30 Inhibitor Protein Im9 bound to its partner E9 DNase
1IMQ 1997-07-07 COLICIN E9 IMMUNITY PROTEIN IM9, NMR, MINIMIZED AVERAGE STRUCTURE
2VLQ 2008-05-20 1.6 F86A mutant of E9 DNase domain in complex with Im9
2GZJ 2008-03-18 1.6 Crystal Structure of the E9 DNase Domain with a Mutant Immunity Protein IM9 (D51A)
1FR2 2003-06-17 1.6 CRYSTAL STRUCTURE OF THE E9 DNASE DOMAIN WITH A MUTANT IMMUNITY PROTEIN IM9(E41A)
2GYK 2007-05-15 1.6 Crystal structure of the complex of the Colicin E9 DNase domain with a mutant immunity protein, IMME9 (D51A)
2VLN 2008-05-20 1.6 N75A mutant of E9 DNase domain in complex with Im9
2GZI 2008-03-18 1.7 Crystal Structure of the E9 DNase Domain with a Mutant Immunity Protein IM9 (V34A)
2GZG 2008-03-18 1.7 Crystal Structure of the E9 DNase Domain with a Mutant Immunity Protein IM9 (Y55F)
1EMV 2000-09-13 1.7 CRYSTAL STRUCTURE OF COLICIN E9 DNASE DOMAIN WITH ITS COGNATE IMMUNITY PROTEIN IM9 (1.7 ANGSTROMS)
2GZF 2007-07-03 1.75 Crystal structure of the E9 DNase domain with a mutant immunity protein IM9 (Y54F)
2GZE 2008-03-18 1.8 Crystal structure of the E9 DNase domain with a mutant immunity protein IM9 (Y55A)
2VLO 2008-05-20 1.8 K97A mutant of E9 DNase domain in complex with Im9
2VLP 2008-05-20 2.0 R54A mutant of E9 DNase domain in complex with Im9
1BXI 1999-10-04 2.05 CRYSTAL STRUCTURE OF THE ESCHERICHIA COLI COLICIN E9 DNASE DOMAIN WITH ITS COGNATE IMMUNITY PROTEIN IM9
3GKL 2009-09-08 2.2 Following evolutionary paths to high affinity and selectivity protein-protein interactions using Colicin7 and Immunity proteins
3GJN 2009-09-15 2.48 Following evolutionary paths to high affinity and selectivity protein-protein interactions using Colicin7 and Immunity proteins
5EW5 2016-07-20 3.2 Crystal Structure of Colicin E9 In Complex with Its Immunity Protein Im9

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Colicin-E9 immunity protein P13479 IMM9_ECOLX