Structure,dynamics, and ionization equilibria of the tyrosine residues in <Emphasis Type="Italic">Bacillus circulans</Emphasis> xylanase

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 ³J_CH coupling between the ring ¹H^η and ¹³C^ε 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 ¹H^η 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 ¹³C^ζ chemical shifts via ¹H^ε/δ(¹³C^ε)¹³C^ζ 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.