Measurement of bond vector orientations in invisible excited states of proteins

Pramodh Vallurupalli, D. Flemming Hansen, Elliott Stollar, Eva Meirovitch, Lewis E. Kay

Research output: Contribution to journalArticlepeer-review

161 Scopus citations

Abstract

The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where 1H-15N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.

Original languageEnglish
Pages (from-to)18473-18477
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number47
DOIs
StatePublished - 20 Nov 2007

Keywords

  • CPMG
  • Chemical exchange
  • Dipolar couplings
  • Dynamics
  • NMR

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