Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and (19)F NMR spectroscopy

J Am Chem Soc. 2002 Feb 27;124(8):1778-81. doi: 10.1021/ja016748e.

Abstract

Oxygen solubility increases toward the hydrophobic interior of membranes. Using NMR, this O(2) solubility gradient gives rise to an exquisite range of position-dependent paramagnetic effects at partial pressures of 100 atm (PO(2)), which may be used to probe membrane protein structure and positioning. In this study, fluorinated probes were introduced at selected positions of the transmembrane 1 domain of the intact homotrimer of the integral membrane protein, diacylglycerol kinase. Using (19)F NMR, O(2)-induced chemical shift perturbations revealed secondary structure, membrane immersion depth, and regions of the helix in contact with the protein or with the micelle.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cysteine / chemistry
  • Cysteine / genetics
  • Diacylglycerol Kinase / chemistry*
  • Diacylglycerol Kinase / genetics
  • Fluorine
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular / methods
  • Oxygen / chemistry*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary

Substances

  • Membrane Proteins
  • Fluorine
  • Diacylglycerol Kinase
  • Cysteine
  • Oxygen