Utilizing tagged paramagnetic shift reagents to monitor protein dynamics by NMR

Publication date: Available online 22 September 2017
Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Author(s): Libin Ye, Ned van Eps, Xiang Li, Oliver P. Ernst, R. Scott Prosser
Calmodulin is a ubiquitous calcium sensor protein, known to serve as a critical interaction hub with a wide range of signaling partners. While the holo form of calmodulin (CaM-4Ca²⁺) has a well-defined ground state structure, it has been shown to undergo exchange, on a millisecond timescale, to a conformation resembling that of the peptide bound state. Tagged paramagnetic relaxation agents have been previously used to identify long-range dipolar interactions through relaxation effects on nuclear spins of interest. In the case of calmodulin, this lead to the determination of the relative orientation of the N- and C-terminal domains and the presence of a weakly populated peptide bound like state. Here, we make use of pseudocontact shifts from a tagged paramagnetic shift reagent which allows us to define minor states both in ¹³C and ¹⁵N NMR spectra and through ¹³C- and ¹⁵N-edited ¹H-CPMG relaxation dispersion measurements. This is validated by pulsed EPR (DEER) spectroscopy which reveals an ensemble consisting of a compact peptide-bound like conformer, an intermediate peptide-bound like conformer, and a (dumbbell-like) extended ground state conformer of CaM-4Ca²⁺, where addition of the MLCK peptide increases the population of the peptide-bound conformers. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.



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