Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins

Lindsey J. Brown, Matthias Baranowski, Yun Wang, Anna K. Schrey, Thomas Lenz, Sean Dixon Taverna, Philip A. Cole, Michael Sefkow

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)14-21
Number of pages8
JournalAnalytical Biochemistry
Volume467
DOIs
StatePublished - Dec 15 2014

Fingerprint

S-Adenosylmethionine
Homocysteine
Carrier Proteins
Proteins
Fluorescence Polarization
Streptavidin
Methyltransferases
Horseradish Peroxidase
Biotin
Fluorescein
Crosslinking
Lysine
Tumors
Anisotropy
Fluorescence

Keywords

  • Capture compound
  • Fluorescence anisotropy
  • S-Adenosyl-L-homocysteine
  • S-Adenosyl-L-methionine

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins. / Brown, Lindsey J.; Baranowski, Matthias; Wang, Yun; Schrey, Anna K.; Lenz, Thomas; Taverna, Sean Dixon; Cole, Philip A.; Sefkow, Michael.

In: Analytical Biochemistry, Vol. 467, 15.12.2014, p. 14-21.

Research output: Contribution to journalArticle

Brown, Lindsey J. ; Baranowski, Matthias ; Wang, Yun ; Schrey, Anna K. ; Lenz, Thomas ; Taverna, Sean Dixon ; 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. In: Analytical Biochemistry. 2014 ; Vol. 467. pp. 14-21.
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