Graduate Student
Medford, Massachusetts, Us
NMR and X-Ray based study of two facets of serine protease function: the pKa of Asp102 and the origin of the characteristic low-field 1H-NMR peak. Despite decades of research, the pKa of the acidic member of the catalytic triad has gone undefined, until now. This work is published - See Below.The presence of low-field 1H-NMR resonances in serine protease spectra has been acknowledged for years and have been a crucial component in many method-of-action studies and mechanistic studies. We discovered that the low-field resonances in alpha-lytic protease were due entirely to the presence of autocatalytic degradants formed during lyophilization, a procedure deemed 100% necessary to observe the signals at all. These degradants were identified as 5- and 7-mer peptides which, as poor substrates for alpha, contorted the active site into producing these aberration in a mechanistically important way.SKILLS = Protein expression in bacterial/mammalian-plasmid expression systems • High yield HPLC/FPLC-based protein purification (Akta Purifier w/ IEX, RP, affinity, filtration) • 1D/2D/3D NMR including 1H, 15N, 13C, RDC, HCACO, HCCH-TOCSY • X-Ray Crystallography (crystallization trials, structure refinement, solution = 3QGJ.pdb) • MS-based protease-peptide stability assays (LC/MS, Agilent 1100/Thermo LCQ Duo) • Kinetic parameter determination (Km, Kcat, IC50, Ki) • In vitro inhibition studies with boronic acids, peptide mimetics, and other site-directed inhibitors • Mammalian tissue culture • Lyophilization
