Analysis of p300 acetyltransferase substrate specificity by MALDI TOF mass spectrometry.
Acetyltransferase enzymes target specific lysine residues in substrate proteins. While the list of histone and nonhistone substrates is growing, the mechanisms of substrate selection remain unclear. Here, we describe a mass spectrometric approach to examine the site selection of the acetyltransferase p300 in the HIV-1 protein Tat. Tat is acetylated by p300 at a single lysine (K50) within its basic RNA-binding domain. To determine the sequence requirements for K50 recognition within this domain, we synthesized mixtures of "degenerated" Tat peptides, in which one of the surrounding residues was substituted by all proteinogenic amino acids. Peptide mixtures were assembled based on nonoverlapping peptide masses and acetylated by p300 in a standard in vitro acetylation reaction. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified amino acid substitutions that prevented acetylation by p300. This approach represents a fast and comprehensive screening method that was applied to the six surrounding residues of K50 in Tat. It can be applied to any known acetyltransferase substrate and might help to define consensus recognition sequences for individual acetyltransferase enzymes.
Dormeyer
Wilma
W
Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany.
Ott
Melanie
M
Schnölzer
Martina
M
D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D016384:Consensus Sequence / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D051548:Histone Acetyltransferases; D006801:Humans; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D018797:Cell Cycle Proteins; D015696:Gene Products, tat; D051548:Histone Acetyltransferases; D010446:Peptide Fragments; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor