Whenever macromolecules change their behavior or properties as a consequence of proximity to another macromolecule, interactions are at play.
Reversible complexes are formed as a result of non-covalent interactions producing dynamic equilibrium between complexes and constituent monomers. This equilibrium is concentration-dependent, so a fractionation technique such as SEC-MALS - with the accompanying time-dependent dilution - is not suitable for full and rigorous characterization of reversible complexes. Among the most useful techniques for studying reversible complexes is composition-gradient light scattering (CG-MALS).
CG-MALS characterizes self- and hetero-association.
Non-covalent interactions generally fall into two classes:
- Specific interactions – those that lead to formation of well-defined complexes consisting of exact molecular stoichiometries (e.g., protein-ligand complexes).
- Non-specific interactions – those due to the hard-core repulsion, electrostatic repulsion and/or attraction, weak hydrophobic attraction, van der Waals potentials, etc. The net effect of these interactions may be repulsive or attractive; while they may lead to the formation of complexes or loosely-bound aggregates, these typically do not exhibit a well-defined stoichiometry or oligomeric state.
CG-MALS is adept at characterizing both specific and non-specific interactions to determine the magnitude of the interaction and the true molecular stoichiometry (when applicable) of resultant complexes and oligomers. While not as sensitive or rigorous, Dynamic Light Scattering (DLS) offers microwell-plate-based, high-throughput screening of interactions and their dependence on buffer conditions.
CG-MALS works by mixing different compositions of samples and diluents, then measuring the weight-average molar mass of the solution at each composition step. Binding kinetics are often apparent.