Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes.
Acta Crystallogr Sect F Struct Biol Cryst Commun
; 68(Pt 5): 596-600, 2012 May 01.
Article
in En
| MEDLINE
| ID: mdl-22691796
ABSTRACT
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.
Full text:
1
Database:
MEDLINE
Main subject:
Pyridoxal Phosphate
/
Bacterial Proteins
/
Fluorometry
/
Transaminases
Language:
En
Journal:
Acta Crystallogr Sect F Struct Biol Cryst Commun
Year:
2012
Type:
Article
Affiliation country:
United States