title: Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9.
abstract: The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: S A
authors.initials: SA
authors.lastname: Gorski
authors.forename: A P
authors.initials: AP
authors.lastname: Capaldi
authors.forename: C
authors.initials: C
authors.lastname: Kleanthous
authors.forename: S E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000143:Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003087:Colicins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: D000143:Acids; D001426:Bacterial Proteins; D003087:Colicins; C007104:colicin immunity proteins; D014508:Urea