Structure, dynamics, and ionization equilibria of the tyrosine residues in Bacillus circulans 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 (13)C-HSQC experiment that exploits the (3)J(CH) coupling between the ring (1)H(η) and (13)C(ε) nuclei. The NMR signals from these protons were assigned via (13)C-tyrosine selective labelling and a suite of scalar and (13)C,(15)N-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 (13)C-(1)H 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 (13)C longitudinal exchange experiment. The hydrogen bonding properties of the Tyr79 and Tyr105 hydroxyls were also defined by complementary NOE and J-coupling measurements. The (1)H(η) hydrogen-deuterium exchange rate constants of the buried tyrosines were determined from (13)C/(15)N-filtered spectra recorded as a function of pH. These exchange rate constants correspond to estimated protection factors of ~10(4)-10(8) relative to a random coil tyrosine. The phenolic sidechain pK (a) values were also measured by monitoring their pH-dependent (13)C(ζ) chemical shifts via (1)H(ε/δ)((13)C(ε))(13)C(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.