Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

EF-hand Ca-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca. Upon binding Ca, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca-binding affinity of CaM and most S100s in the absence of target is weak (K > 1 μM). However, upon effector protein binding, the Ca affinity of these proteins increases via heterotropic allostery (K < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca homeostasis and (ii) strict maintenance of Ca-signaling within a narrow dynamic range of free Ca ion concentrations, [Ca]. In this review, mechanisms of allostery are coalesced into an empirical "binding and functional folding (BFF)" physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.