The choice of which molecule to keep constant and which to vary will depend on the experiment type, the properties of the molecules and the stoichiometry of the interaction.
For ITC, where neither protein is labeled or immobilized, either molecule can be in the cell or syringe. You will need a higher concentration and total amount of the molecule in the syringe, so solubility and availability will generally be deciding factors. For protein/small molecule interactions, it is most common to put the protein in the cell and the compound in the syringe.
The relative size of the molecules isn't generally the deciding factor. Large proteins will typically give larger signals, but that isn’t necessarily better.
For protein/protein interactions, start by considering other properties of the proteins. Most importantly stoichiometry of binding. If one of the molecules is multivalent (an antibody, for example),...
