Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data

Fatrick J. Farber, Jelle Slager, Anthony K. Mittermaier*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

16 Citations (Scopus)

Abstract

NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments represent a powerful approach for characterizing protein internal motions and for gaining insight into fundamental biological processes such as protein folding, catalysis, and allostery. In most cases, CPMG data are analyzed assuming that the protein exchanges between two different conformational states. Systems exchanging among more than two states are far more challenging to characterize by CPMG NMR. For example, in the case of three-state exchange in the fast time scale regime, it is difficult to uniquely connect the parameters extracted from CPMG analyses with the physical parameters of most interest, intercoversion rates, populations, and chemical shift differences for exchanging states. We have developed a grid search selection procedure that allows these physical parameters to be uniquely determined from CPMG data, based on additional information, which in this study comprises ligand-induced chemical shift perturbations. We applied this approach to the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminal extension that folds into a fourth helix upon binding to DNA. We recently showed that the C-terminal extension transiently folds, even in the absence DNA, in a process that is likely tied to the cooperative binding of PBX-HD to DNA and other homeodomains. Using the grid search selection procedure, we found that PBX-HD undergoes exchange between three different conformational states, a major form in which the C-terminal extension is unfolded, the previously identified state in which the C-terminal extension forms a fourth helix, and an additional state in which the C-terminal extension is misfolded.

Original languageEnglish
Pages (from-to)10317-10329
Number of pages13
JournalJournal of Physical Chemistry B
Volume116
Issue number34
DOIs
Publication statusPublished - 30-Aug-2012

Keywords

  • NUCLEAR-MAGNETIC-RESONANCE
  • COOPERATIVE DNA-BINDING
  • TIME-SCALE DYNAMICS
  • CHEMICAL-SHIFT ANISOTROPY
  • SH3 DOMAIN
  • HOX PROTEINS
  • BIOLOGICAL MACROMOLECULES
  • BACKBONE DYNAMICS
  • EXCHANGE
  • SPECTROSCOPY

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