posted on 2024-01-31, 08:54authored byClaris
Niya Varghese, Ahallya Jaladeep, Ashok Sekhar
Molecular
recognition events mediated by glycans play pivotal roles
in controlling the fate of diverse biological processes such as cellular
communication and the immune response. The affinity of glycans for
their target receptors is governed primarily by the hydrogen bonds
formed by hydroxyl groups decorating the glycan surface. Hydroxyl
exchange rate constants are therefore vital parameters that report
on glycan structure and dynamics. Here we present a strategy for characterizing
hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs
a synergistic combination of 13C chemical exchange saturation
transfer (CEST) and Carr–Purcell–Meiboom–Gill
relaxation dispersion (CPMG) NMR methods. We show that the combination
of CEST and CPMG experiments facilitates the sensitive detection of
the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between
protonated and deuterated states. This shift is leveraged for measuring
site-specific kinetic H/D exchange rate constants as well as thermodynamic
free energies of isotope fractionation. The CEST and CPMG modules
are integrated with a selective J-cross-polarization scheme that provides
the flexibility for rapid characterization of H/D exchange at a specific
hydroxyl site. Moreover, our approach enables the precise isothermal
measurement of hydroxyl exchange rate constants without the need for
cumbersome isotope labeling. The H/D exchange rate constants of three
different glycans assessed using this method highlight its potential
for detecting transient intra- and intermolecular hydrogen bonds.
In addition, the trends in H/D exchange rate constants establish site-specific
steric accessibility as a key determinant of solvent exchange dynamics
in glycans.