2014
Brown, Lindsey J; Baranowski, Matthias; Wang, Yun; Schrey, Anna K; Lenz, Thomas; Taverna, Sean D; Cole, Philip A; Sefkow, Michael
Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins Journal Article
In: Anal Biochem, vol. 467, pp. 14-21, 2014, ISSN: 1096-0309.
Abstract | Links | BibTeX | Tags: Catechol O-Methyltransferase, DNA-Binding Proteins, Fluorescence Polarization, Histone-Lysine N-Methyltransferase, Humans, Hydrolases, Nuclear Proteins, S-Adenosylhomocysteine, S-Adenosylmethionine, Transcription Factors
@article{1299978,
title = {Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins},
author = {Lindsey J Brown and Matthias Baranowski and Yun Wang and Anna K Schrey and Thomas Lenz and Sean D Taverna and Philip A Cole and Michael Sefkow},
doi = {10.1016/j.ab.2014.08.013},
issn = {1096-0309},
year = {2014},
date = {2014-12-01},
journal = {Anal Biochem},
volume = {467},
pages = {14-21},
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.},
keywords = {Catechol O-Methyltransferase, DNA-Binding Proteins, Fluorescence Polarization, Histone-Lysine N-Methyltransferase, Humans, Hydrolases, Nuclear Proteins, S-Adenosylhomocysteine, S-Adenosylmethionine, Transcription Factors},
pubstate = {published},
tppubtype = {article}
}
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.