Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies

Abstract

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.

Keywords: IDP; biophysics; coupled folding and binding; electrostatics; kinetics; phi-value; protein dynamic; protein electrostatics; protein folding; protein-protein interactions; residual structure; signaling.

FIGURE 1.

The thermodynamic and kinetic properties of IDPs vary over orders of magnitude, and may be related to their function. Examples are given from studies in our laboratory (21, 24, 31).

FIGURE 2.

Kinetic experiments of coupled folding and binding reactions under pseudo-first order conditions. A, example kinetic traces for two-state and three-state processes, fit to single exponential and double exponential decay functions, respectively. a. u., arbitrary units. B, dependence of observed rate constants upon protein concentrations. Analytical solutions are presented for a two-state reaction (k_on = 40 μm⁻¹ s⁻¹, k_off = 10 s⁻¹) and a three-state IF reaction (k₊ = 40 μm⁻¹ s⁻¹, k₋ = 10 s⁻¹, k_f = 10 s⁻¹, k_u = 20 s⁻¹). C, reaction schemes for two-state and three-state (IF and CS) processes.

FIGURE 3.

Relationship between association and dissociation rate constants and φ-values for apparent two-state systems. Shown are energy diagrams (first column), observed association rate constants under pseudo-first order conditions (middle column), and observed dissociation rate constants (third column) for wild-type IDP (blue) and mutant IDP (red). A, φ = 1, i.e. native interactions are formed in the transition state. k_on is lower, and k_off is unchanged. B, 0 < φ <1 structure is partially formed, resulting in changes in both k_on and k_off. C, φ = 0, residue is as unstructured at the transition state as in the unbound state. k_on is unchanged, and k_off is increased. The rate constants k_on and k_off are controlled by energy barrier sizes (first column), and are determined from straight-line gradients in association mixing experiments (second column) and from high concentration asymptotes in out-competition dissociation mixing experiments (third column), respectively.

FIGURE 4.

Illustrations of coupled folding and binding reaction transition states. φ-Values are mapped onto structures of the following complexes: A, PUMA·Mcl-1 (PDB: 2ROC) (43); B, c-Myb·CBP KIX (PDB: 1SBO) (51); C, S-peptide·S-protein (PDB: 1FEV) (55); D, ACTR·NCBD (PDB: 1KBH) (; E, α·β spectrin tetramerization domain (PDB: 3LBX) (44). In A, B, and C, the folded partners are shown in gray. In D and E, both partners are disordered; one is shown in gray, and one is shown in bronze. The residues in blue, magenta, and red represent high (φ > 0.6), medium (0.25 ≥ φ ≤0.6), and low (φ < 0.25) φ-values, respectively. N and C denote the N and C termini of the IDP (note that in E the disordered regions are capped by folded domains).