Synthesis and characterization of a fluorescent analogue of cyclic di-GMP.
Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Indra Mani
IM
Sharma
Thillaivillalan
T
Dhanaraman
Ritta
R
Mathew
Dipankar
D
Chatterji
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D060326:Chemistry Techniques, Synthetic; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D015203:Reproducibility of Results
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP