Abstract
Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.
Highlights
Internal cavities are important elements in protein structure, dynamics, stability and function
Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein
Many crystal structures of heme-proteins with O2 ligands and their migration processes inside the proteins have been investigated by X-ray crystallography[22,23] and molecular dynamics (MD) simulation[24,25], to the best of our knowledge, association of O2 with internal cavities of proteins in solution has been investigated by nuclear magnetic resonance (NMR) spectroscopy only for ribonuclease A12,26, deoxymyoglobin[13], and the B domain of protein A20
Summary
Internal cavities are important elements in protein structure, dynamics, stability and function. Internal cavities in proteins are important structural elements that may produce functional motions[1], such as drug and ligand binding[2] and conformational transitions into high-energy states[3,4,5,6,7] To explore their locations and dynamic aspects, specific binding of noble gases, xenon, into protein cavities has been studied by X-ray crystallography[8,9,10]. X-ray crystallography suggested that the enlarged cavity in L99A is sterically inaccessible to incoming ligands, NMR spin relaxation studies showed the presence of conformational fluctuations around the hydrophobic cavities and the rapid exchange of benzene and indole with the protein interior[30,31,32,33,34]. Our objective here is to understand the selectivity of O2 to hydrophilic and hydrophobic cavities and the coupling between protein conformational fluctuation and accessibility of O2 to internal cavities of the protein
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