Enzymatic activation complex formation induced by crowding stress

Calmodulin (CaM), a calcium-sensing protein, regulates several important biological signaling pathways, including the catalytic production of nitric oxide (NO) by the nitric oxide synthase (NOS) enzyme. It is generally accepted that 4 Ca²⁺-bound CaM, the activated CaM, is associated with NOS enzymes and activates NO biosynthesis. Recent studies have shown that molecular crowding has a significant impact on protein dynamics and function, as the cell interior contains a large number of macromolecules. Herein, we use single-molecule fluorescence resonance energy transfer (smFRET) analysis to study the effects of the macromolecule on the CaM molecule dynamics, CaM-enzyme complex formations, and CaM-activated enzymatic reaction pathways. We have studied the CaM-eNOS complex formation in the presence of the macromolecule Ficoll 70 as a molecular crowder, without the addition of CaCl₂. Our results indicate that macromolecules significantly impact the association, dissociation, folding, and unfolding of protein molecules, which may result in several pathways in protein signaling and function in the human body. We have demonstrated that chemical pathways do not solely govern the formation of the CaM-eNOS complex, but that macromolecular crowding also plays a significant role in the formation of an active CaM-eNOS complex. Our results suggest that CaM signaling and NOS functions can be regulated with the combined action of chemical-induced and force-induced pathways, providing new insights into the force-induced pathways for the NOS enzymatic reactions.