The importance of dynamic light scattering in obtaining multiple crystal forms of Trypanosoma brucei PGK.
Phosphoglycerate kinase (PGK) catalyzes the phosphoryl transfer between 1,3 bis-phosphoglycerate and ADP to form 3-phosphoglycerate and ATP, undergoing significant conformational changes during catalysis. To more precisely document this reaction and the corresponding conformational changes, we have crystallized Trypanosoma brucei PGK in several crystal forms: (1) in the presence of 3-phosphoglycerate and MgADP, PGK crystallizes with four molecules in the asymmetric unit; (2) in the presence of the ATP analog, AMP-PNP, PGK crystallizes in a similar form; (3) in the presence of the bisubstrate analog, adenylyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate, PGK crystals grow with one molecule in the asymmetric unit. Large scale expression and purification of T. brucei PGK from an E. coli overexpression system was required to obtain sufficient enzyme yields. Results from dynamic light scattering experiments allowed us to identify substrates and analogs which were amenable for crystallization. Ease of crystal growth and diffraction quality for a particular PGK-ligand complex is highly consistent with the apparent monodispersity of the complex in solution as judged by dynamic light scattering. The three-dimensional structures of the various enzyme-ligand complexes are currently being exploited to obtain a better understanding of PGK catalysis, as well as for structure based design of enzyme inhibitors to be used in the development of anti-trypanosomal agents.
Bernstein
B E
BE
Department of Biochemistry, Howard Hughes Medical Institute, University of Washington, Seattle 98195, USA.
Michels
P A
PA
Kim
H
H
Petra
P H
PH
Hol
W G
WG
D000818:Animals; D018360:Crystallography, X-Ray; D008027:Light; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase