title: Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins.
abstract: S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.
authors.affiliation: Center for Epigenetics, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Lindsey J
authors.initials: LJ
authors.lastname: Brown
authors.affiliation: Caprotec Bioanalytics, 12489 Berlin, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Baranowski
authors.affiliation: Center for Epigenetics, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Caprotec Bioanalytics, 12489 Berlin, Germany.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Schrey
authors.affiliation: Caprotec Bioanalytics, 12489 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lenz
authors.affiliation: Center for Epigenetics, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Taverna
authors.affiliation: Center for Epigenetics, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
authors.affiliation: Caprotec Bioanalytics, 12489 Berlin, Germany. Electronic address: michael.sefkow@caprotec.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Sefkow
mesh_terms: D002394:Catechol O-Methyltransferase / Q000032:analysis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D011495:Histone-Lysine N-Methyltransferase / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000032:analysis* / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D011495:Histone-Lysine N-Methyltransferase; C093563:PRDM2 protein, human; C588010:COMT protein, human; D002394:Catechol O-Methyltransferase; D006867:Hydrolases; C019614:adenosylmethionine hydrolase
keywords: Capture compound; Fluorescence anisotropy; S-Adenosyl-l-homocysteine; S-Adenosyl-l-methionine