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Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

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Abstract

Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca2+)4-form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2–3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca2+ to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca2+ binding. The midpoint of the Ca2+-mediated transition determined by EPR occurred at a higher Ca2+ concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca2+)4-CaM], contains a compact intermediate corresponding to [(Ca2+)2-CaM], and the present results suggest that the spin probes are reporting on Ca2+ binding to the last two sites in the N-terminal domain, i.e. for the [(Ca2+)2-CaM] → [(Ca2+)4-CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca2+-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.

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Abbreviations

CaM:

Calmodulin

CD:

Circular dichroism

EGTA:

Ethylene glycol-bis(2-aminoethylether-N,N,N,N-tetraacetic acid

EPR:

Electron paramagnetic resonance

HMQC:

Heteronuclear multiple quantum coherence

mW:

Milliwatts

NMR:

Nuclear magnetic resonance

PDE:

Phosphodiesterase (cyclic nucleotide)

SEM:

Standard error of the mean

SL-CaM:

Spin-labeled calmodulin

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Acknowledgments

We are most appreciative of the helpful comments, suggestions, and assistance from Professors Malcolm Forbes (University of North Carolina), William Lanzilotta (University of Georgia), John Rose (University of Georgia), Alex Smirnov (North Carolina State University), and Zachary Wood (University of Georgia). We also thank Professor Albert Beth (Vanderbilt University) for helpful discussions and for obtaining EPR spectra of precipitated spin-labeled calmodulin. Lastly, a special thanks to Mr. Edward Larry Bowman for his invaluable assistance in preparing the figures and to Mr. Connor Puett for his expert help. This research was supported by the National Institutes of Health (Grants DK33973, GM35415, GM07319, and RR05424).

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The authors declare no conflict of interest.

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The experiments presented herein comply with the current laws of the USA.

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Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA

    Paula B. Bowman

  2. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    David Puett

  3. Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, 27599, USA

    David Puett

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  1. Paula B. Bowman
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Correspondence to David Puett.

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Paula B. Bowman was formerly known as Paula B. Hewgley.

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Bowman, P.B., Puett, D. Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin. Protein J 33, 267–277 (2014). https://doi.org/10.1007/s10930-014-9559-9

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