Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes

Todd W. Geders, Kathryn Gustafson, Barry C. Finzel

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)596-600
Number of pages5
JournalActa Crystallographica Section F: Structural Biology and Crystallization Communications
Volume68
Issue number5
DOIs
StatePublished - May 2012

Keywords

  • Crystallization optimization
  • Differential scanning fluorimetry
  • Pyridoxal 5'-phosphate
  • ThermoFluor
  • UV-Vis spectroscopy

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