Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

Abstract

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.hydrophobic cavity ͉ molecular movie ͉ protein dynamics ͉ time-resolved crystallography L ocalized electronic excitation by photons often induces largescale structural modulations and novel physical properties in condensed matter (1, 2). Myoglobin (Mb), often referred to as the hydrogen atom of biology and a paradigm of complexity (3), has played a central role in research on the photo-induced response of proteins and migration of gases, solvents, and ligands in the protein matrix (3, 4). Despite the large number of details known about Mb dynamics, it remains unclear how a ligand molecule escapes from the protein matrix to the solvent and how the protein matrix responds to the ligand migration at the atomic level. A number of time-resolved spectroscopic measurements of Mb photoproducts have revealed a complex ligand-binding reaction with multiple kinetic intermediates (4-8). After dissociation from the heme iron atom, ligand gas molecules either rebind internally from the distal pocket (DP) (Fig. 1) or escape into the solvent. It has been deduced that the escape of the ligand is assisted by the thermal f luctuations that transiently open exit channels. Lowering the temperature slows down the thermal f luctuations, and the internal binding process becomes dominant (4, 5).The multiple kinetic intermediates scheme of Mb has motivated researchers to characterize the structural features of the the intermediates by using both time-resolved (9-14) and cryogenic crystallographic measurements (15)(16)(17)(18)(19)(20)(21)(22). A general picture emerging from these experiments is that Mb...

Control of the Position of Oxygen Delivery in Soybean Lipoxygenase-1 by Amino Acid Side Chains within a Gas Migration Channel

“…In 12/15 lipoxygenase, a preferred route for oxygen travel has been identified that links the solvent and buried active site (6). Similarly, crystallographic and computational studies have also implicated oxygen access paths in cyclooxygenase, copper amine oxidase, cholesterol oxidase, and cytochrome c oxidase (8,10,11). The nuanced role of the protein matrix in influencing catalysis is not restricted to oxygen delivery.…”

Blocking the Gate to Ligand Entry in Human Hemoglobin

“…Many of these pathways are coincident with apolar cavities, which have been identified as xenon docking sites (16, 17, 28 -30). However, as also pointed out by Elber (11), there is little or no direct experimental evidence in support of multiple pathways (31,32). In the cases of mammalian myoglobins, human hemoglobin, and HbI from Scapharca inaequivalvis, almost all of the experimental evidence suggests that Ն75% of ligands enter and exit the distal pocket through a transient channel between the heme propionates, which is produced by outward rotation of the distal histidine (His(E7)).…”

Strain analysis of protein structures and low dimensionality of mechanical allosteric couplings

“…Yamato (27) calculated strain tensor fields induced by high pressure in lysozyme and myglobin. Recently, Yamato and coworkers have applied a similar, molecular dynamics-based approach to supplement their analyses of protein dynamics (13,28).…”

“…12), timeresolved crystallography (e.g., ref. 13), FRET (e.g., ref. 14), and direct pulling measurements (15,16) have been published.…”