caa a22 4500 445325240 CHVBK 20180317142718.0 cr unu---uuuuu 170323e20111101xx s 000 0 eng 10.1007/s10858-011-9564-7 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9564-7 Structure, dynamics, and ionization equilibria of the tyrosine residues in Bacillus circulans xylanase [Elektronische Daten] [Simon Baturin, Mark Okon, Lawrence McIntosh] 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 12.5ppm 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 ~0.2s−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. Springer Science+Business Media B.V., 2011 Glycoside hydrolase nationallicence Tyrosine hydroxyl nationallicence Protein dynamics nationallicence Protein electrostatics nationallicence NMR spectroscopy nationallicence Baturin Simon Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Okon Mark Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut McIntosh Lawrence Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut Journal of Biomolecular NMR Springer Netherlands 51/3(2011-11-01), 379-394 0925-2738 51:3<379 2011 51 10858 https://doi.org/10.1007/s10858-011-9564-7 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9564-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Baturin Simon Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- Okon Mark Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 700 1- McIntosh Lawrence Department of Biochemistry and Molecular Biology, Department of Chemistry, and Michael Smith Laboratories, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/3(2011-11-01), 379-394 0925-2738 51:3<379 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer