Perrone, B. | Search bioRxiv

bioRxiv2023

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

biophysics↗

bioRxiv2021

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography - the synergistic combination of solid-state NMR, X-ray crystallography, and first-principles computational chemistry - holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in the enzyme active site - including hydrogen atom locations and tautomeric equilibria - it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the -aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the {beta}-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the {beta}-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate {beta}-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the -aminoacrylate C{beta} and aligned for nucleophilic attack. Significance StatementThe determination of active site protonation states is critical to gaining a full mechanistic understanding of enzymatic transformations; yet hydrogen positions are challenging to extract using the standard tools of structural biology. Here we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that unlocks the investigation of enzyme catalytic mechanism at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the -aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to proton positions - rivaling that of neutron diffraction, yet able to be applied under conditions of active catalysis to microcrystalline and non-crystalline materials.

biophysics↗