Cooperative binding are binding events in which the Binding Affinity of a molecule to an interaction partner is influenced by a preceding binding event. There are two kinds of cooperative binding.

Homotropic cooperativity means that the binding of one molecule A to a molecule B increases or decreases the affinity of B for further molecules of A. The most famous example of homotropic cooperativity is oxygen binding to hemoglobin: after the first oxygen molecule is bound to one heme unit, the oxygen affinity of the other heme units increases to facilitate complete loading of hemoglobin with four oxygen molecules. Homotropic cooperativity of course always comes with a non-1:1 binding stoichiometry, which can be analyzed using Spectral Shift or TRIC.

Heterotrophic cooperativity on the other hand, refers to a change in affinity of the interaction of molecules A and B that is brought about by the binding of molecule C. An example for this is synaptotagmin-1, a calcium sensor. It can bind both Ca²⁺ and phosphatidylinositol 4,5-bisphosphate (PIP2). Binding of PIP2 to synaptotagmin-1 increases the affinity of the sensor protein to Ca²⁺. Heterotrophic cooperativity in this example comes with a 1:1:1 stoichiometry, but other models (and more interaction partners) are possible. In GPCRs, heterotrophic cooperativity between different binding sites is often referred to as allosteric modulation. To investigate heterotrophic cooperativity, analyze the affinity of A to B in the presence of various concentrations of C.

An increasing affinity upon binding is called positive cooperativity, while a decreasing affinity is called negative cooperativity. When investigating interactions that are known to exhibit homotropic cooperativity, use the Hill Model for fitting the binding curve. In all other, including unknown, cases, please use the Kd Fit Model.

Note: The Hill coefficient does not describe the stoichiometry of an interaction but rather it's cooperativity. In general, when working with Hill coefficients one should be cautious with their interpretation.