Functional significance of hierarchical tiers in carbonmonoxy myoglobin: Conformational substates and transitions studied by conformational flooding simulations

The functional importance of large-scale motions and transitions of carbonm onoxy myoglobin (MbCO) conformational substates (Cs) has been studied by mo lecular dynamics (MD) and conformational flooding (CF) simulations. A flood ing potential was constructed from an 800 ps MD trajectory of solvated MbCO to accelerate slower protein motions beyond the time scale of contemporary simulations. Two conformational transitions (tier-l substates) resulting f rom seven principal molecular motions were assigned to the spectroscopic Ao state (tier-0 substate) of MbCO, where His64 is solvated and nor within th e hydrophobic pecker binding site. The first computed conformational transi tion involves a distal pocket gate defined by the C and D helices and the i nterconnecting CD loop (residues 40-55). The gate-like motion is interprete d to regulate ligand access from the distal side of the hems. Simultaneousl y, a proximal pocket lever involving the F helix and surrounding EF and FG loops (residues 82-105) is found to shuttle the heme deep into the protein matrix (heme rmsd of 3.9 Angstrom) as the distal pocket gate opened. The le ver's effect on the heme motion is assumed to attract ligands into the heme pocket. The second major transition involves the compression and expansion of the cavity formed by the EF loop (residues 77-84) and the GH loop and H helix (residues 122-138). The motion is interpreted to modulate the hydrop hobic pocket volume and regulate the ligand access from the proximal side o f the heme. A third computed conformational transition was Found to be a co mbination of the previous motions. For the first time, CF was applied in a series of room temperature simulations to accelerate molecular motions of t he MbCO native fold and define the lower tier hierarchy of substate structu re. The computed CSs and associated transitions coincide with previously su ggested putative ligand escape pathways, and support a hierarchical descrip tion of protein dynamics and structure. A unified model that utilizes both mechanisms of distal His64 modulation (tier-0) and protein equilibrium fluc tuations (tier-l) is presented to explain ligand diffusion in the MbCO diss ociation reaction.