A mass spectrometry‐based non‐radioactive differential radial capillary action of ligand assay (DRaCALA) to assess ligand binding to proteins. (1st April 2022)

• Record Type: Journal Article
• Authors: Cimdins‐Ahne, Annika; Chernobrovkin, Alexey; Kim, Soo‐Kyoung; Lee, Vincent T.; Zubarev, Roman A.; Römling, Ute
• Is Part Of: Journal of mass spectrometry. Volume 57:Number 4(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-04-01
• Subjects: biofilm -- cyclic di‐GMP -- diguanylate cyclase/phosphodiesterase -- mass spectrometry -- non‐radioactive DRaCALA; Mass spectrometry -- Periodicals; 543.65
• DOI: 10.1002/jms.4822
• Languages: English
• ISSNs: 1076-5174

Abstract

Abstract: Binding of ligands to macromolecules changes their physicochemical and enzymatic characteristics. Cyclic di‐GMP is a second messenger involved in motility/sessility and acute/chronic infection life style transition. Although the GGDEF domain, predominantly a diguanylate cyclase, represents one of the most abundant bacterial domain superfamilies, the number of cyclic di‐GMP receptors falls short. To facilitate screening for cyclic di‐nucleotide binding proteins, we describe a non‐radioactive, matrix‐assisted laser desorption and ionization time‐of‐flight (MALDI‐TOF)‐based modification of the widely applied differential radial capillary action of ligand assay (DRaCALA). The results of this assay suggest that the diguanylate cyclase/phosphodiesterase variant YciRFec101, but not selected catalytic mutants, bind cyclic di‐GMP. Highlights: Cyclic di‐nucleotides are ubiquitous second messengers in bacteria. However, few receptors have been identified. Previous screening of cell lysates by differential radial capillary action of ligand assay (DRaCALA) using radioactive ligand identified cyclic di‐nucleotide binding proteins. A MALDI‐TOF‐based DRaCALA was developed to detect cyclic di‐nucleotide binding as a non‐radioactive alternative. Known cyclic di‐GMP binding proteins were verified and potential cyclic di‐GMP binding proteins were identified.