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Approaches to the assignment of 19F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR

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Abstract

Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ 15N and 19F-edited two- and three-dimensional NMR experiments for purposes of assigning 19F NMR resonances from calmodulin (CaM), biosynthetically labeled with 3-fluorophenylalanine (3-FPhe). The global substitution of 3-FPhe for native phenylalanine was tolerated in CaM as evidenced by a comparison of 1H-15N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The 19F NMR spectrum reveals six resolved resonances, one of which integrates to three 3-FPhe species, making for a total of eight fluorophenylalanines. Single phenylalanine to tyrosine mutants of five phenylalanine positions resulted in 19F NMR spectra with significant chemical shift perturbations of the remaining resonances, and provided only a single definitive assignment. Although 1H-19F heteronucleclear NOEs proved weak, 19F-edited 1H-1H NOESY connectivities were relatively easy to establish by making use of the 3JFH coupling between the fluorine nucleus and the adjacent fluorophenylalanine δ proton. 19F-edited NOESY connectivities between the δ protons and α and β nuclei in addition to 15N-edited 1H, 1H NOESY crosspeaks proved sufficient to assign 4 of 8 19F resonances. Controlled cleavage of the protein into two fragments using trypsin, and a repetition of the above 2D and 3D techniques resulted in unambiguous assignments of all 8 19F NMR resonances. Our studies suggest that 19F-edited NOESY NMR spectra are generally adequate for complete assignment without the need to resort to mutational analysis.

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Abbreviations

1D:

One-dimensional

2D:

Two-dimensional

3D:

Three-dimensional

CaM:

Calmodulin

CSA:

Chemical shift anisotropy

DNase:

Deoxyribonuclease

RNase:

Ribonuclease

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

EDTA:

Ethylenediaminetetraacetic acid

NMR:

Nuclear magnetic resonance

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Acknowledgments

We wish to thank Professor Mitsu Ikura (University of Toronto) for providing the plasmid for Xenopus laevis calmodulin. Michael Bokoch (Stanford University) for the preparation of phenylalanine to tyrosine mutant DNA. We would like to acknowledge Professors Lewis Kay and Julie Forman-Kay (University of Toronto) for helpful comments. Julianne Kitevski-LeBlanc wishes to acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for a doctoral fellowship and RSP acknowledges NSERC and the Canadian Foundation for Innovation (CFI) for financial support through the NSERC discovery program and CFI New Opportunities programs.

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Authors and Affiliations

  1. Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON, L5L 1C6, Canada

    Julianne L. Kitevski-LeBlanc, Ferenc Evanics & R. Scott Prosser

  2. Department of Biochemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada

    R. Scott Prosser

Authors
  1. Julianne L. Kitevski-LeBlanc
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  2. Ferenc Evanics
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  3. R. Scott Prosser
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Correspondence to R. Scott Prosser.

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Kitevski-LeBlanc, J.L., Evanics, F. & Scott Prosser, R. Approaches to the assignment of 19F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR. J Biomol NMR 47, 113–123 (2010). https://doi.org/10.1007/s10858-010-9415-y

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