Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis.
The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (KD) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse.
Institute of Pharmacy and Molecular Biotechnology; Heidelberg University; Im Neuenheimer Feld 364; Heidelberg, Germany.
Ece Cazibe
EC
Gaffarogullari
Institute of Pharmacy and Molecular Biotechnology; Heidelberg University; Im Neuenheimer Feld 364; Heidelberg, Germany.
André
A
Krause
Cellnetworks Cluster and Institute for Physical Chemistry; Heidelberg University; Im Neuenheimer Feld 267; Heidelberg, Germany.
Jessica
J
Balbo
Cellnetworks Cluster and Institute for Physical Chemistry; Heidelberg University; Im Neuenheimer Feld 267; Heidelberg, Germany.
Dirk-Peter
DP
Herten
Institute of Pharmacy and Molecular Biotechnology; Heidelberg University; Im Neuenheimer Feld 364; Heidelberg, Germany.
Andres
A
Jäschke
D000873:Anthracenes / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D061565:Cycloaddition Reaction; D008301:Maleimides / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D000873:Anthracenes; C518747:Diels-Alder ribozyme; D008301:Maleimides; D016337:RNA, Catalytic
Diels-Alder reaction; fluorescence correlation spectroscopy; microscale thermophoresis; ribozyme; thermodynamics