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 Kd 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.
Highlights
The association and dissociation of protein complexes is of enormous biological significance, especially because most proteins exist in such forms in vivo, with monomers accounting for only 19% of proteins in Escherichia coli [1]
For one intrinsically disordered proteins (IDPs) system, that estimates for the association and dissociation rate constants, and for the equilibrium dissociation constant for the complex, can be extracted from individual kinetic traces fitted to an equation appropriate for a twostate reversible process
We found that the kinetic traces were well fit by Eq 5, which contains only four free variables; DF, F0, kþ, and Kd, without the requirement for further fixing
Summary
The association and dissociation of protein complexes is of enormous biological significance, especially because most proteins exist in such forms in vivo, with monomers accounting for only 19% of proteins in Escherichia coli [1]. More recently, there has been increasing interest in association of intrinsically disordered proteins (IDPs), proteins that are largely unstructured in the unbound state and that form structure only upon binding another protein [2,3,4]. These coupled folding and binding reactions are predicted to be widespread in biology [5], with important examples found in signaling [3], transcription [6], apoptosis regulation [7], and disease-related proteins [8].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
