Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes.
Differential scanning fluorimetry (DSF) is a practical and accessible technique that allows the assessment of multiphasic unfolding behavior resulting from subsaturating binding of ligands. Multiphasic unfolding is indicative of a heterogenous protein solution, which frequently interferes with crystallization and complicates functional characterization of proteins of interest. Along with UV-Vis spectroscopy, DSF was used to guide purification and crystallization improvements for the pyridoxal 5'-phosphate (PLP) dependent transaminase BioA from Mycobacterium tuberculosis. The incompatibility of the primary amine-containing buffer 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) and PLP was identified as a significant contributor to heterogeneity. It is likely that the utility of DSF for ligand-binding assessment is not limited to the cofactor PLP but will be applicable to a variety of ligand-dependent enzymes.
Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Todd W
TW
Geders
Kathryn
K
Gustafson
Barry C
BC
Finzel
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D005470:Fluorometry / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D011732:Pyridoxal Phosphate; D000637:Transaminases; C102100:BioA protein, bacteria