 Solution

A: downward when ligand is present; B: downward; C: upward, over the intended protein and ligand concentration range. Ligand should favour its bound Y state, and the ligand-bound subunits should favour tetramer assembly. Without ligand, the unassembled state should predominate, rather than forming a stable ligand-free tetramer.

These preferences are thermodynamically coupled. The ligand must bind preferentially to states participating in the assembled structure strongly enough to compensate for the cost of shifting the unliganded conformational and assembly equilibria. At extremely low protein concentration, association entropy can still favour monomers; “ligand-dependent” does not mean concentration-independent assembly. This is a thermodynamic linkage argument, rather than three independently adjustable arrows.