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Specificity Analysis of Protein Methyltransferases and Discovery of Novel Substrates Using SPOT Peptide Arrays.
Weirich, Sara; Jeltsch, Albert.
Afiliação
  • Weirich S; Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
  • Jeltsch A; Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany. albert.jeltsch@ibtb.uni-stuttgart.de.
Methods Mol Biol ; 2529: 313-325, 2022.
Article em En | MEDLINE | ID: mdl-35733022
ABSTRACT
Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substrate sequence, yielding a substrate sequence specificity profile. This information can then be used to identify novel potential PMT substrates by in silico data base searches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Peptídeos / Proteínas Metiltransferases Tipo de estudo: Prognostic_studies Idioma: En Revista: Methods Mol Biol Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2022 Tipo de documento: Article País de afiliação: Alemanha

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Peptídeos / Proteínas Metiltransferases Tipo de estudo: Prognostic_studies Idioma: En Revista: Methods Mol Biol Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2022 Tipo de documento: Article País de afiliação: Alemanha
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