Utilizing tagged paramagnetic shift reagents to monitor protein dynamics by NMR

作者：Ye Libin; Van Eps Ned; Li Xiang; Ernst Oliver P; Prosser R Scott^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2017, 1865(11): 1555-1563.

DOI：10.1016/j.bbapap.2017.09.011

• F-19 NMR
• Calmodulin
• Conformational dynamics
• Paramagnetic shifts
• CPMG
• DEER
• Pulsed EPR

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 ealmodulin (CaM-4Ca(2+)) 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 C-13 and N-15 NMR spectra and through C-13- and N-15 -edited H-1-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-4Ca(2 +), 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.