Molecular oxygen as a paramagnetic NMR probe of protein solvent exposure and topology

Bezsonova, Irina; Forman‐Kay, Julie D.; Prosser, R. Scott

doi:10.1002/cmr.a.20118

Cited by 14 publications

(13 citation statements)

References 39 publications

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“…Penetration of dissolved oxygen (O 2 ) into proteins was originally investigated by quenching of fluorescence 16 17 . The paramagnetic effects of O 2 , such as paramagnetic shifts and paramagnetic relaxation enhancements (PREs), have been used to study protein solvent exposure and topology by NMR spectroscopy 18 19 20 21 . Although many crystal structures of heme-proteins with O 2 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 O 2 with internal cavities of proteins in solution has been investigated by NMR spectroscopy only for ribonuclease A 12 26 , deoxymyoglobin 13 , and the B domain of protein A 20 .…”

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confidence: 99%

Detecting O2 binding sites in protein cavities

Kitahara

Yoshimura

Xue

et al. 2016

Sci Rep

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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.

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confidence: 99%

Detecting O2 binding sites in protein cavities

Kitahara

Yoshimura

Xue

et al. 2016

Sci Rep

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“…If the pseudo-contact shift dominated, the magnitudes of chemical shift changes for the bound nuclei would be to a similar degree in ppm. However, similarity of O 2 -induced chemical shifts among 1 H, 13 C, and 15 N nuclei were not observed. More details surrounding this observation were previously discussed.…”

Section: Dissociation Constants Of Omentioning

confidence: 82%

“…NMR provides a high‐resolution approach to investigate the structure and dynamics of proteins in solution. Several studies have shown that NMR spectroscopy in combination with dioxygen (O 2 ) gas‐pressure is a powerful tool for investigating the location of internal protein cavities and pockets in solution and their accessibility to ligands, owing to the paramagnetic properties of O 2 . In previous studies, methods to analyze O 2 ‐binding sites in proteins in solution were reported, using the O 2 ‐induced enhancement of backbone amide proton spin‐lattice relaxation .…”

Section: Introductionmentioning

confidence: 99%

“…Several studies have shown that NMR spectroscopy in combination with dioxygen (O 2 ) gaspressure is a powerful tool for investigating the location of internal protein cavities and pockets in solution and their accessibility to ligands, owing to the paramagnetic properties of O 2 . [12][13][14][15][16][17][18] In previous studies, methods to analyze O 2 -binding sites in proteins in solution were reported, using the O 2 -induced enhancement of backbone amide proton spin-lattice relaxation. 12,15,16 We found that O 2 bound selectively to the hydrophobic, rather than hydrophilic (water-containing) cavities in the cavity-enlarged T4 lysozyme mutant L99A.…”

Section: Introductionmentioning

confidence: 99%

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Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

Kitahara

Sakuraba

Kameda³

et al. 2018

Protein Science

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The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O in solvent to 8.9 mM, O -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O binding sites and their atomic coordinates from the 1/r distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O revealed dioxygen ingress and egress as well as rotational and translational motions of O in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O association with the internal protein cavities.

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“…Oxygen presents distinct advantages as a sPRE probe owing to its small size and ubiquitous nature. However, one needs to bear in mind that significant PREs on 1 H and 19 F can only be observed at pressures of 20–60 bar 11. Additionally, analysis can be obscured by the inherent electrostatic bias that predominates the interaction between oxygen and the solute.…”

Section: Spre Probesmentioning

confidence: 99%

Studying the Structure and Dynamics of Biomolecules by Using Soluble Paramagnetic Probes

2013

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Characterisation of the structure and dynamics of large biomolecules and biomolecular complexes by NMR spectroscopy is hampered by increasing overlap and severe broadening of NMR signals. As a consequence, the number of available NMR spectroscopy data is often sparse and new approaches to provide complementary NMR spectroscopy data are needed. Paramagnetic relaxation enhancements (PREs) obtained from inert and soluble paramagnetic probes (solvent PREs) provide detailed quantitative information about the solvent accessibility of NMR-active nuclei. Solvent PREs can be easily measured without modification of the biomolecule; are sensitive to molecular structure and dynamics; and are therefore becoming increasingly powerful for the study of biomolecules, such as proteins, nucleic acids, ligands and their complexes in solution. In this Minireview, we give an overview of the available solvent PRE probes and discuss their applications for structural and dynamic characterisation of biomolecules and biomolecular complexes.

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Molecular oxygen as a paramagnetic NMR probe of protein solvent exposure and topology

Cited by 14 publications

References 39 publications

Detecting O2 binding sites in protein cavities

Detecting O2 binding sites in protein cavities

Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

Studying the Structure and Dynamics of Biomolecules by Using Soluble Paramagnetic Probes

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