Analysis of CheA histidine phosphorylation and its influence on protein stability by high-resolution element and electrospray mass spectrometry.
A combination of electrospray mass spectrometry (ESI-MS) and element mass spectrometry (ICPMS) with phosphorus detection was used to characterize histidine phosphorylation (His-48) of the chemotaxis protein CheA quantitatively. The phosphorylation at His-48 was found to be responsible for a stabilization of the protein. For this investigation, the acceptor domain and the kinase domain of the bacterial chemotaxis protein CheA were recombinantly expressed as single proteins. Using in vitro kinase assay conditions, the acceptor domain CheA-H was phosphorylated by the kinase domain CheA-C. The degree of histidine phosphorylation was determined by nanoelectrospray mass spectrometry of intact CheA-H, and was found to be limited to a maximum value of approximately 50%. The site specificity of CheA-H phosphorylation was controlled by nanoESI-MS/MS of the [M + 16H](16+) ion of intact (pHis)-CheA-H and allowed localization of the pHis residue to the region between residues 32 and 86, containing candidates His-48 and His-67, for which His-48 phosphorylation has been described. Analysis of the tryptic digest of in vitro histidine-phosphorylated CheA-H by capillary chromatography coupled to ESI-MS and to ICPMS with phosphorus detection revealed a truncated (pHis)-CheA-H protein as the only phosphorus-containing analyte. Since the truncated (pHis)-CheA-H in the digest was found to exhibit a higher degree of phosphorylation than could be generated by in vitro phosphorylation without trypsin treatment, it is concluded that histidine phosphorylation at His-48 strongly interferes with structural properties of the CheA-H domain in particular with respect to proteolytic degradation by trypsin.
Wind
Mathias
M
Central Spectroscopy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Wegener
Ansgar
A
Kellner
Roland
R
Lehmann
Wolf D
WD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D006639:Histidine / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011994:Recombinant Proteins; D006639:Histidine