title: Slow, reversible, coupled folding and binding of the spectrin tetramerization domain.
abstract: Many intrinsically disordered proteins (IDPs) are significantly unstructured under physiological conditions. A number of these IDPs have been shown to undergo coupled folding and binding reactions whereby they can gain structure upon association with an appropriate partner protein. In general, these systems display weaker binding affinities than do systems with association between completely structured domains, with micromolar K(d) values appearing typical. One such system is the association between α- and β-spectrin, where two partially structured, incomplete domains associate to form a fully structured, three-helix bundle, the spectrin tetramerization domain. Here, we use this model system to demonstrate a method for fitting association and dissociation kinetic traces where, using typical biophysical concentrations, the association reactions are expected to be highly reversible. We elucidate the unusually slow, two-state kinetics of spectrin assembly in solution. The advantages of studying kinetics in this regime include the potential for gaining equilibrium constants as well as rate constants, and for performing experiments with low protein concentrations. We suggest that this approach would be particularly appropriate for high-throughput mutational analysis of two-state reversible binding processes.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: S L
authors.initials: SL
authors.lastname: Shammas
authors.forename: J M
authors.initials: JM
authors.lastname: Rogers
authors.forename: S A
authors.initials: SA
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Clarke
mesh_terms: D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013049:Spectrin / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
chemical_list: D013049:Spectrin; D014364:Tryptophan; D014508:Urea