Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Abstract

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

Keywords: coupling energy; interaction networks; site-directed mutagenesis.

Figure 1

Schematic representation of a double mutant cycle. P-XY is the wild-type protein with the two residues X and Y. P-X and P-Y are the variants where Y and X are mutated, respectively, and P is the corresponding double mutant. The change in free energy upon mutation of X (or Y) is equal to ΔΔG_P-XY→P-Y (or ΔΔG_P-XY→P-X) and ΔΔG_P-XY→P is the change in free energy of the double mutant.

Figure 2

Structural distribution of residues L18, I26, L43, Y65, L67, and Y73 (green spheres) in the KIX domain (PDB 2AGH). The analysis of folding m values, in combination with double mutant cycle analysis, allowed to characterize, from a thermodynamic perspective (i.e., measuring ΔΔΔG values), the role of these residues in stabilizing non-native interactions in the denatured state of KIX. With the exception of I26 and L43 that are in direct contact, residues displaying significant coupling free energies are located in different regions of the domain (see [28] for details).

Figure 3

Graphic representation of the different allosteric networks of PDZ domain-ligand complexes of protein tyrosine phosphatase Basophil-like PDZ2 (a and c panels) and PSD-95 PDZ3 (b and d panels) with their natural ligand (showed below each panel). All the mutated residues are represented as spheres both in the ligand (blue) and in the PDZ domain (red). The peptide ligand mutations Val→Abu (deletion of a γ-methyl group from Val), Ser→Thr or Thr→Ser were chosen because they interact with the binding pocket of PDZ domains.