title: The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9.
abstract: How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Phi-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Phi-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Morton
authors.forename: Claire T
authors.initials: CT
authors.lastname: Friel
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Allen
authors.forename: Emanuele
authors.initials: E
authors.lastname: Paci
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins