Computationally-guided tuning of ligand sensitivity in a GPCR-based sensor

Genetically-encoded fluorescent sensors for neuromodulators are increasingly used molecular tools in neuroscience. However, these protein-based biosensors are often limited by the sensitivity of the protein scaffold towards endogenous ligands. Here, we explored the possibility of applying computational design approaches for enhancing sensor sensitivity. Using the dopamine sensor dLight1 as proof of concept, we designed two variants that boost the sensor's potency (EC50) for dopamine and norepinephrine by up to 5- and 15-fold, respectively. Interestingly, the largest effects were obtained through improved designed allosteric transmission in the transmembrane region of the sensor. Our approach should prove generally useful for enhancing sensing capabilities of a large variety of neuromodulator sensors.

The Quantity of G-Quadruplex in Bacterial Genome affects G-Quadruplex Ligand Sensitivity in Hypertension associated Tongue Coating Microbiota

Background Recently, more and more attention has been paid to the role of oral microbiota in hypertension. It was reported that the disorder of tongue coating microbiota would significantly increase systolic blood pressure, yet the characteristics of tongue coating microbiota of hypertensives remain unknown. Microbiota is regulated by genes, G-quadruplex (G4) is a secondary structure of nucleic acid, which plays an important role in the regulation of microbial biologic features. there are many G4 ligands in oral administration, but how G4 ligands affect the bacterial biological phenotype needs further exploration. Results We used metagenomics for analysis of 58 subjects including 23 healthy subjects and 35 hypertensives, and bioinformatics technology for detecting G4 characteristic sequences, finally verified by biological and chemical experiments. We found that Actinomyces decreased significantly in the hypertension group with the highest average maximum putative G-quadruplex forming sequences (PQS) and GC quantity. We also screened out two species with significantly different abundance between two groups, Actinomyces odontolyticus and Acinetobacter baumannii. A. odontolyticus had higher GC% and frequency of PQS per 1000bp in the genome, which led to differential inhibition of bacterial growth, metabolism, biofilm formation by G4 ligand, sanguinarine. Major Facilitator Superfamily (MFS) was found to be involved in these biological phenomena, we found that sanguinarine could bind and stabilize the G4 structure related to MFS and further inhibited the expression of MFS.ConclusionsDifferent quantities of G4 sequences in the bacterial genome affect G4 ligand sensitivity in hypertension-associated tongue coating species, A. odontolyticus and A. baumannii. G4 ligand (sanguinarine) can bind and stabilize the G4 characteristic sequence of the MFS gene to inhibit the expression, and then inhibit the growth, biofilm formation and metabolism. This study provides a theoretical basis for the selection of G4 based drugs in the oral cavity.

Binding and structural asymmetry governs ligand sensitivity in a cyclic nucleotide–gated ion channel

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open more easily when cAMP or cGMP bind to a domain in the intracellular C-terminus in each of four identical subunits. How sensitivity of the channels to these ligands is determined is not well understood. Here, we apply a mathematical model, which incorporates negative cooperativity, to gating and mutagenesis data available in the literature and combine the results with binding data collected using isothermal titration calorimetry. This model recapitulates the concentration–response data for the effects of cAMP and cGMP on wild-type HCN2 channel opening and, remarkably, predicts the concentration–response data for a subset of mutants with single-point amino acid substitutions in the binding site. Our results suggest that ligand sensitivity is determined by negative cooperativity and asymmetric effects on structure and channel opening, which are tuned by ligand-specific interactions and residues within the binding site.