Oxygen as a paramagnetic probe of clustering and solvent exposure in folded and unfolded states of an SH3 domain

Irina Bezsonova, Ferenc Evanics, Joseph A Marsh, Julie D Forman-Kay, R Scott Prosser

Research output: Contribution to journalArticlepeer-review

Abstract

The N-terminal SH3 domain of the Drosophila modular protein Drk undergoes slow exchange between a folded (Fexch) and highly populated unfolded (Uexch) state under nondenaturing buffer conditions, enabling both Fexch and Uexch states to be simultaneously monitored. The addition of dissolved oxygen, equilibrated to a partial pressure of either 30 atm or 60 atm, provides the means to study solvent exposure with atomic resolution via 13C NMR paramagnetic shifts in 1H,13C HSQC (heteronuclear single quantum coherence) spectra. Absolute differences in these paramagnetic shifts between the Fexch and Uexch states allow the discrimination of regions of the protein which undergo change in solvent exposure upon unfolding. Contact with dissolved oxygen for both the Fexch and Uexch states could also be assessed through 13C paramagnetic shifts which were normalized based on the corresponding paramagnetic shifts seen in the free amino acids. In the Fexch state, the 13C nuclei belonging to the hydrophobic core of the protein exhibited very weak normalized paramagnetic shifts while those with greater solvent accessible surface area exhibited significantly larger normalized shifts. The Uexch state displayed less varied 13C paramagnetic shifts although distinct regions of protection from solvent exposure could be identified by a lack of such shifts. These regions, which included Phe9, Thr12, Ala13, Lys21, Thr22, Ile24, Ile27, and Arg38, overlapped with those found to have residual nativelike and non-native structures in previous studies and in some cases provided novel information. Thus, the paramagnetic shifts from dissolved oxygen are highly useful in the study of a transient structure or clustering in disordered systems, where conventional NMR measurements (couplings, chemical shift deviations from random coil values, and NOEs) may give little information.
Original languageEnglish
Pages (from-to)1826-35
Number of pages10
JournalJournal of the American Chemical Society
Volume129
Issue number6
DOIs
Publication statusPublished - 14 Feb 2007

Keywords

  • Carbon Isotopes
  • Drosophila Proteins
  • Electron Spin Resonance Spectroscopy
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Oxygen
  • Protein Folding
  • src Homology Domains

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