Time filter

Source Type

Buck J.,Institute for Organic Chemistry and Chemical Biology | Wacker A.,Institute for Organic Chemistry and Chemical Biology | Warkentin E.,Max Planck Institute of Biophysics | Wohnert J.,Goethe University Frankfurt | And 2 more authors.
Nucleic Acids Research | Year: 2011

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution. © The Author(s) 2011. Published by Oxford University Press.

Wacker A.,Institute for Organic Chemistry and Chemical Biology | Buck J.,Institute for Organic Chemistry and Chemical Biology | Mathieu D.,Institute for Organic Chemistry and Chemical Biology | Richter C.,Institute for Organic Chemistry and Chemical Biology | And 2 more authors.
Nucleic Acids Research | Year: 2011

The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 20-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purinesensing riboswitches. We show that the mflaptamer accommodates the extra 20-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xptaptamer bound to guanine and of the mfl-aptamer bound to 20-deoxyguanosine exhibit very similar features, although the sequence of the mflaptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg 2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer. © 2011 The Author(s).

Leuner K.,Goethe University Frankfurt | Heiser J.H.,Goethe University Frankfurt | Derksen S.,Institute for Organic Chemistry and Chemical Biology | Mladenov M.I.,University of Heidelberg | And 9 more authors.
Molecular Pharmacology | Year: 2010

The naturally occurring acylated phloroglucinol derivative hyperforin was recently identified as the first specific canonical transient receptor potential-6 (TRPC6) activator. Hyperforin is the major antidepressant component of St. John's wort, which mediates its antidepressant-like properties via TRPC6 channel activation. However, its pharmacophore moiety for activating TRPC6 channels is unknown. We hypothesized that the phloroglucinol moiety could be the essential pharmacophore of hyperforin and that its activity profile could be due to structural similarities with diacylglycerol (DAG), an endogenous nonselective activator of TRPC3, TRPC6, and TRPC7. Accordingly, a few 2-acyl and 2,4-diacylphloroglucinols were tested for their hyperforin-like activity profiles. We used a battery of experimental models to investigate all functional aspects of TRPC6 activation, including ion channel recordings, Ca2+ imaging, neurite outgrowth, and inhibition of synaptosomal uptake. Phloroglucinol itself was inactive in all of our assays, which was also the case for 2-acylphloroglucinols. For TRPC6 activation, the presence of two symmetrically acyl-substitutions with appropriate alkyl chains in the phloroglucinol moiety seems to be an essential prerequisite. Potencies of these compounds in all assays were comparable with that of hyperforin for activating the TRPC6 channel. Finally, using structure-based modeling techniques, we suggest a binding mode for hyperforin to TRPC6. Based on this modeling approach, we propose that DAG is able to activate TRPC3, TRPC6, and TRPC7 because of higher flexibility within the chemical structure of DAG compared with the rather rigid structures of hyperforin and the 2,4-diacylphloroglucinol derivatives. Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics.

Thevarpadam J.,Goethe University Frankfurt | Bessi I.,Institute for Organic Chemistry and Chemical Biology | Binas O.,Institute for Organic Chemistry and Chemical Biology | Goncalves D.P.N.,Goethe University Frankfurt | And 6 more authors.
Angewandte Chemie - International Edition | Year: 2016

The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two-tetrad G-quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G-quadruplexes with K+ ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para-para-substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after E-Z isomerization. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA.

Discover hidden collaborations