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Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy

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Abstract

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of 1H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond 1H–15N or 1H–13C couplings. However, by filtering against protons directly bonded to 13C or 15N nuclei, signals from slowly-exchanging hydroxyls can be observed in the 1H-NMR spectrum of a uniformly 13C/15N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with 13C/15N-serine or 13C/15N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via 3JC–OH couplings in long-range 13C-HSQC spectra. These scalar couplings (~5–7 Hz) were measured in a sample of uniformly 13C/15N-labeled BcX using a quantitative 13C/15N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a 13C/15N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

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Acknowledgments

We thank Simon Baturin for help with preliminary experiments. This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) to LPM. Instrument support was provided by the Canadian Institutes for Health Research (CIHR), the Canada Foundation for Innovation (CFI), the British Columbia Knowledge Development Fund (BCKDF), the UBC Blusson Fund, and the Michael Smith Foundation for Health Research (MSFHR).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Life Sciences Centre, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada

    Jacob A. Brockerman, Mark Okon & Lawrence P. McIntosh

  2. Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

    Jacob A. Brockerman, Mark Okon & Lawrence P. McIntosh

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  1. Jacob A. Brockerman
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  2. Mark Okon
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  3. Lawrence P. McIntosh
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Correspondence to Lawrence P. McIntosh.

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Brockerman, J.A., Okon, M. & McIntosh, L.P. Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy. J Biomol NMR 58, 17–25 (2014). https://doi.org/10.1007/s10858-013-9799-6

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