Hideyuki Yamaguchi

Monitoring the hydrolysis and transglycosylation activity of α-glucosidase from Aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry

Alpha-glucosidase from Aspergillus niger is an enzyme that catalyzes hydrolysis of alpha-1,4 linkages and transglucosylation to form alpha-1,6 linkages. In this study, an analytical method of oligosaccharides by nuclear magnetic resonance (NMR) was used to provide quantitative estimation of the fractions of each sugar unit and was applied to characterize the alpha-glucosidase reaction. Our data indicated that alpha-glucosidase reacts with the nonreducing end of oligosaccharides to form an alpha-1,6 linkage, and then a sugar unit with two alpha-1,6 linkages is gradually produced. Data from mass spectrometry suggested that the sugar unit with two alpha-1,6 linkages originates mainly from a 3mer and/or 4mer when oligosaccharides are used as substrates.

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.