Determination of Leu side-chain conformations in excited protein states by NMR relaxation dispersion

D Flemming Hansen, Philipp Neudecker, Pramodh Vallurupalli, Frans A A Mulder, Lewis E Kay

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

Abstract

Fits of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion profiles allow extraction of the kinetics and thermodynamics of exchange reactions that interconvert highly populated, ground state and low populated, excited state conformers. Structural information is also available in the form of chemical shift differences between the interconverting protein states. Here we present a very simple method for extracting chi(2) rotamer distributions of Leu side chains in 'invisible' excited protein states based on measurement of their (13)C(delta1)/(13)C(delta2) chemical shifts using methyl CPMG dispersion experiments. The methodology is applied to study the protein folding reaction of the Fyn SH3 domain. A uniform chi(2) rotamer distribution is obtained for Leu residues of the unfolded state, with each Leu occupying the trans and gauche+ conformations in a 2:1 ratio. By contrast, leucines of an 'invisible' Fyn SH3 domain folding intermediate show a much more heterogeneous distribution of chi(2) rotamer populations. The experiment provides an important tool toward the quantitative characterization of both the structural and dynamics properties of states that cannot be studied by other biophysical tools.

Original languageEnglish
JournalJournal of the American Chemical Society
Volume132
Issue1
Pages (from-to)42-43
Number of pages2
ISSN0002-7863
DOIs
Publication statusPublished - 13 Jan 2010
Externally publishedYes

Keywords

  • Leucine
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Protein Conformation
  • Proto-Oncogene Proteins c-fyn
  • Stereoisomerism
  • src Homology Domains

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