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

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Ryo Kitahara, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-8577, Japan.
  • ,
  • Shun Sakuraba, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan.
  • ,
  • Tomoshi Kameda, Artificial Intelligence Research Center, Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto, Tokyo 135-0064, Japan.
  • ,
  • Sanshiro Okuda, Graduate School of Life Sciences, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-8577, Japan.
  • ,
  • Mengjun Xue
  • ,
  • Frans A A Mulder

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -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 O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -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 O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities. This article is protected by copyright. All rights reserved.

Original languageEnglish
JournalProtein Science
Volume27
Issue3
Pages (from-to)769-779
Number of pages11
ISSN0961-8368
DOIs
Publication statusPublished - 1 Mar 2018

    Research areas

  • Journal Article

See relations at Aarhus University Citationformats

ID: 120254838