Chaperonin assisted overexpression, purification, and characterisation of human PP2A methyltransferase.
Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase found in many eukaryotic cell types and is involved in regulating a number of intracellular signalling pathways. Its activity, in turn, is regulated through covalent modification, involving phosphorylation and methylation reactions. The effect of phosphorylation on the activity of the protein is well known, but the effects of methylation have only recently been documented and the mechanistic details of methylation are lacking. Methylation, which occurs on the catalytic subunit of PP2A, is catalysed by PP2A methyltransferase (PP2Amt). Here, we present a method for the large-scale purification of human PP2Amt using an Escherichia coli host, coexpressing the chaperonins GroEL and GroES. Purified PP2Amt was identified by peptide mass mapping using MALD-MS and peptide sequencing using ESI-LC-MS/MS. The CD spectrum indicated that purified PP2Amt was folded, with about one-third of the protein adopting an alpha-helical conformation. Analytical gel filtration estimated the molecular weight to be 34kDa, equivalent to the monomeric form of the protein. Further CD analysis showed that in the presence and absence of the ligand S-adenosylhomocysteine, the thermal denaturation profiles were biphasic. However, the transition midpoints shifted to a higher temperature in the presence of ligand, indicating stabilisation of ligand-bound PP2Amt compared to the apo-form. We also report on the progress made in determining the structure of PP2Amt, using both X-ray crystallography and NMR spectroscopy.
George
Roger R
RR
Department of Biochemistry and Molecular Biology, University College London, London, WC1E 6BT, UK.
Harris
Richard
R
Nunn
Christine M
CM
Cramer
Rainer
R
Djordjevic
Snezana
S
D001483:Base Sequence; D018833:Chaperonins / Q000502:physiology*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011496:Protein Methyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D011496:Protein Methyltransferases; D018833:Chaperonins