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G-quadruplex conformation and dynamics are determined by loop length and sequence.

ABSTRACT: The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.

SUBMITTER: Tippana R

PROVIDER: S-EPMC4081081 | biostudies-other | 2014 Jul

REPOSITORIES: biostudies-other

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G-quadruplex conformation and dynamics are determined by loop length and sequence.

Tippana Ramreddy R Xiao Weikun W Myong Sua S

Nucleic acids research 20140611 12

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that ...[more]

PMID: 24920827

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Project description:Background and purposeTransient receptor potential melastatin 3 (TRPM3) is a non-selective cation channel that plays a pivotal role in the peripheral nervous system as a transducer of painful heat signals. Alternative splicing gives rise to several TRPM3 variants. The functional consequences of these splice isoforms are poorly understood. Here, the pharmacological properties of TRPM3 variants arising from alternative splicing in the pore-forming region were compared.Experimental approachCalcium microfluorimetry and patch clamp recordings were used to compare the properties of heterologously expressed TRPM3α1 (long pore variant) and TRPM3α2-α6 (short pore variants). Furthermore, site-directed mutagenesis was done to investigate the influence of the length of the pore loop on the channel function.Key resultsAll short pore loop TRPM3α variants (TRPM3α2-α6) were activated by the neurosteroid pregnenolone sulphate (PS) and by nifedipine, whereas the long pore loop variant TRPM3α1 was insensitive to either compound. In contrast, TRPM3α1 was robustly activated by clotrimazole, a compound that does not directly activate the short pore variants but potentiates their responses to PS. Clotrimazole-activated TRPM3α1 currents were largely insensitive to established TRPM3α2 antagonists and were only partially inhibited upon activation of the μ opioid receptor. Finally, by creating a set of mutant channels with pore loops of intermediate length, we showed that the length of the pore loop dictates differential channel activation by PS and clotrimazole.Conclusion and implicationsAlternative splicing in the pore-forming region of TRPM3 defines the channel's pharmacological properties, which depend critically on the length of the pore-forming loop.Linked articlesThis article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Dataset Information

G-quadruplex conformation and dynamics are determined by loop length and sequence.

Publications

G-quadruplex conformation and dynamics are determined by loop length and sequence.

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