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Structure, dynamics, and ionization equilibria of the tyrosine residues in Bacillus circulans xylanase

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Abstract

We have developed NMR spectroscopic methods to investigate the tyrosines within Bacillus circulans xylanase (BcX). Four slowly exchanging buried tyrosine hydroxyl protons with chemical shifts between 7.5 and 12.5 ppm were found using a long-range 13C-HSQC experiment that exploits the 3JCH coupling between the ring 1Hη and 13Cε nuclei. The NMR signals from these protons were assigned via 13C-tyrosine selective labelling and a suite of scalar and 13C,15N-filtered/edited NOE correlation spectra. Of the fifteen tyrosines in BcX, only the buried Tyr79 and Tyr105 showed four distinct, rather than two averaged, signals from ring 13C–1H pairs, indicative of slow flipping on the chemical shift timescale. Ring flipping rate constants of ~10 and ~0.2 s−1 were measured for the two residues, respectively, using a 13C longitudinal exchange experiment. The hydrogen bonding properties of the Tyr79 and Tyr105 hydroxyls were also defined by complementary NOE and J-coupling measurements. The 1Hη hydrogen–deuterium exchange rate constants of the buried tyrosines were determined from 13C/15N-filtered spectra recorded as a function of pH. These exchange rate constants correspond to estimated protection factors of ~104–108 relative to a random coil tyrosine. The phenolic sidechain pK a values were also measured by monitoring their pH-dependent 13Cζ chemical shifts via 1Hε/δ(13Cε)13Cζ correlation spectra. Exposed tyrosines had unperturbed pK a values of ~10.2, whereas buried residues remained predominantly neutral at or even above pH 11. Combined with selective isotope labelling, these NMR experiments should prove useful for investigating the structural and electrostatic properties of tyrosines in many interesting proteins.

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Acknowledgments

We thank Lewis Kay for continued advice and NMR pulse sequences, as well as Frans Mulder and Jens Nielsen for helpful discussions. This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) to LPM. SJB acknowledges a UBC Aboriginal Graduate Fellowship. Instrument support was provided by the Canadian Institutes for Health Research (CIHR), the Canadian 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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  1. Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada

    Simon J. Baturin, Mark Okon & Lawrence P. McIntosh

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  1. Simon J. Baturin
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Correspondence to Lawrence P. McIntosh.

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10858_2011_9564_MOESM1_ESM.pdf

Supplementary data, including a table of assigned tyrosine chemical shifts, and Fig. showing the NMR pulse sequences used in this study along with the pH-dependent chemical shifts of free tyrosine and the 1Hδ/ε-monitored titration curves of BcX, can be found in the online version. Supplementary material 1 (PDF 673 kb)

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Baturin, S.J., Okon, M. & McIntosh, L.P. Structure, dynamics, and ionization equilibria of the tyrosine residues in Bacillus circulans xylanase. J Biomol NMR 51, 379–394 (2011). https://doi.org/10.1007/s10858-011-9564-7

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