Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant

Acta Crystallogr F Struct Biol Commun. 2019 Apr 1;75(Pt 4):260-269. doi: 10.1107/S2053230X19001882. Epub 2019 Apr 2.

Abstract

Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.

Keywords: Laue method; large crystals; neutron diffraction; perdeuteration; triose-phosphate isomerase.

MeSH terms

  • Amino Acid Sequence
  • Crystallization
  • Crystallography, X-Ray
  • Deuterium / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Leishmania mexicana / enzymology*
  • Mutant Proteins / chemistry*
  • Neutron Diffraction*
  • Triose-Phosphate Isomerase / chemistry*

Substances

  • Mutant Proteins
  • Deuterium
  • Triose-Phosphate Isomerase

Grant support

This work was funded by Crafoordska Stiftelsen grant . Interreg grant . Carl Tryggers Stiftelse för Vetenskaplig Forskning grant .