Speeding molecular recognition by using the folding funnel: The fly-casting mechanism

Abstract

Protein folding and binding are kindred processes. Many proteins in the cell are unfolded, so folding and function are coupled. This paper investigates how binding kinetics is influenced by the folding of a protein. We find that a relatively unstructured protein molecule can have a greater capture radius for a specific binding site than the folded state with its restricted conformational freedom. In this scenario of binding, the unfolded state binds weakly at a relatively large distance followed by folding as the protein approaches the binding site: the ''fly-casting mechanism.'' We illustrate this scenario with the hypothetical kinetics of binding a single repressor molecule to a DNA site and find that the binding rate can be significantly enhanced over the rate of binding of a fully folded protein.W e often glibly state that a protein must be folded to function. The reasoning underlying this statement is that organizing complex networks of chemical reactions in the cell requires these reactions to be highly specific. This specificity is achieved ultimately by having a high degree of geometrical precision in molecular binding-the famous ''lock and key.'' Geometric precision accompanies the increased rigidity of a biomolecule once it has folded; thus, apparently, folding is required for specific function. It does come as a surprise, then, that many proteins in the cell appear to be unfolded most of the time (see ref. 1 and refs. therein). Several ideas about potential biological advantages of being unfolded have been proposed. For example, the rapid turnover of unfolded proteins because of proteolytic degradation may be required for cell-cycle regulation (2). Thermodynamic arguments have also been made suggesting that coupling folding and binding may allow greater equilibrium distinctions for binding to different sites (3). In this note, we investigate whether too much rigidity may conflict with the need for biomolecules to move during their function and is therefore a kinetic disadvantage. There is much evidence for residual flexibility in the folded state, which must be thought of as having an ensemble of conformations (4). Still, the range of motions allowed in the folded state, as measured by Debye-Waller factors (5), is more restricted than those allowed in an unfolded molecule, thus slowing the exploration of configuration space. Here, we illustrate, by means of a specific example of operator binding to DNA, how the speed of molecular recognition can be enhanced by having folding (necessary for the required specificity) occur during binding rather than before.Folding and binding are kindred phenomena. The similarity of binding and folding is clear at the thermodynamic level, where both processes involve accurately locating molecular fragments with respect to each other, reducing the configurational entropy, and simultaneously lowering the free energy by the exclusion of solvent and formation of hydrogen bonds and salt bridges (6, 7). At the structural level, the similarity between packing patterns between ...

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…Motion within a protein is often necessary to guarantee its biological function and is an important component of binding specificity, as highlighted by the discovery of an increasing number of natively unfolded proteins (proteins that adopt an irregular structure in isolation but undergo a folding transition upon binding of a ligand). [18][19][20] Interest in the otherwise biologically functionless denatured state stems from the fact that it represents the starting point of the protein-folding process; thus, a detailed understanding of both structure and dynamics of this thermodynamic macrostate is essential for a complete description of folding. Minimalist lattice models of proteins and Monte Carlo simulations predict a contraction of the denatured polypeptide chain in good solvent (e.g., high concentrations of denaturant) upon transfer in poor solvents (e.g., aqueous solutions) in cases where the overall attraction between residues dominates.…”

“…Characteristic time scales of the fluorescence fluctuations appear in the fitted form of the AF. In the case of free diffusion of a two-state fluorophore ( Figure 1A with no triplet state) diffusing in a 3D Gaussian excitation volume of widths w xy and w z : (18) where N is the number of particles in the observation volume, τ D is the diffusion time of the particle (τ D = w xy 2 /4D), and ω = w z /w xy is the height-to-diameter ratio of the 3D Gaussian confocal volume. In the presence of a triplet state, or any other type of configuration or process resulting in a change of fluorescence emission, the shape of the AF is changed into: (19) where τ R is the relaxation time of the dynamic process and A is its amplitude.…”

Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains

“…According to the socalled "fly-casting" scenario, disordered proteins may quickly form a high energy complex with the physiological partner, which would be locked in place by the subsequent folding reaction. 14 A potential advantage of the intrinsic disorder would then lie in the increased probability to capture a target ligand, 15 even with only moderate affinity. It is of critical importance to address these issues from a biophysical perspective and clarify the role of disorder in protein-ligand recognition.…”

Inherent flexibility and protein function: The open/closed conformational transition in the N-terminal domain of calmodulin

“…Proteins with relatively large conformational freedom include those in which folding and binding are coupled. 33 The dynamics of conformational transitions between well-defined conformational basins are generally controlled by relatively low probability partially ordered ensembles. The main challenge is to describe the transition state ensembles at the residue level giving a site-specific description of the transition mechanism.…”