Bio21 - Research Publications
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ItemUnravelling the mechanism of neurotensin recognition by neurotensin receptor 1(NATURE PORTFOLIO, 2023-12-09)The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.
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ItemNMR techniques for investigating antimicrobial peptides in model membranes and bacterial cells(Elsevier BV, 2024-04)AMPs are short, mainly cationic membrane-active peptides found in all living organism. They perform diverse roles including signaling and acting as a line of defense against bacterial infections. AMPs have been extensively investigated as templates to facilitate the development of novel antimicrobial therapeutics. Understanding the interplay between these membrane-active peptides and the lipid membranes is considered to be a significant step in elucidating the specific mechanism of action of AMPs against prokaryotic and eukaryotic cells to aid the development of new therapeutics. In this review, we have provided a brief overview of various NMR techniques commonly used for studying AMP structure and AMP-membrane interactions in model membranes and whole cells.
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ItemNo Preview AvailableEncounter Complexes Between the N-terminal of Neurotensin with the Extracellular Loop 2 of the Neurotensin Receptor 1 Steer Neurotensin to the Orthosteric Binding Pocket(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2023-10-15)Neurotensin (NT) is a linear disordered peptide that activates two different class A GPCRs, neurotensin receptor 1 (NTS1) and NTS2. Resolved structures of the complex of the C-terminal fragment of NT, NT8-13, with NTS1 shows the peptide takes a well-defined structure in the bound state. However, the mechanisms underlying NT recognition of NTS1, and the conformational transition of NT upon binding NTS1 is an open question that if answered may aid discovery of highly selective drugs and reveal potential secondary binding sites on the surface of the receptor. Herein we investigated the interactions guiding NT to the orthosteric binding pocket of NTS1 by combining NMR experiments with kinetic analysis of the binding pathway using stopped-flow fluorescence and mutagenesis on both NT and NTS1. We show the presence of transient structures in the middle part of NT that kinetically regulate the binding of NT to NTS1. Moreover, our results indicate that the binding pathway of NT onto NTS1 is mediated via electrostatic interactions between the N-terminal region of NT with the extracellular loop 2 of NTS1. These interactions induce backbone conformational changes in neurotensin similar to the bound-state neurotensin, suggesting that the N-terminal region of NT and these interactions should be considered for development of selective drugs against NTS1.
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ItemThe membrane activity of the antimicrobial peptide caerin 1.1 is pH dependent(CELL PRESS, 2023-03-21)Antimicrobial peptides are an important class of membrane-active peptides that can provide alternatives or complements to classic antibiotics. Among the many classes of AMPs, the histidine-rich family is of particular interest since they may induce pH-sensitive interactions with cell membranes. The AMP caerin 1.1 (Cae-1), from Australian tree frogs, has three histidine residues, and thus we studied the pH dependence of its interactions with model cell membranes. Using NMR spectroscopy and molecular dynamics simulations, we showed that Cae-1 induced greater perturbation of the lipid dynamics and water penetrations within the membrane interior in an acidic environment compared with physiological conditions. Using 31P solid-state NMR, the packing, chemical environment, and dynamics of the lipid headgroup were monitored. 2H solid-state NMR showed that Cae-1 ordered the acyl chains of the hydrophobic core of the bilayer. These results supported the molecular dynamics data, which showed that Cae-1 was mainly inserted within the lipid bilayer for both neutral and negatively charged membranes, with the charged residues pulling the water and phosphate groups inward. This could be an early step in the mechanism of membrane disruption by histidine-rich antimicrobial peptides and indicated that Cae-1 acts via a transmembrane mechanism in bilayers of neutral and anionic phospholipid membranes, especially in acidic conditions.
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ItemUtilizing magnetic resonance techniques to study membrane interactions of amyloid peptides(PORTLAND PRESS LTD, 2021-06)Alzheimer's disease (AD) is a common neurodegenerative condition that involves the extracellular accumulation of amyloid plaques predominantly consisting of Aβ peptide aggregates. The amyloid plaques and soluble oligomeric species of Aβ are believed to be the major cause of synaptic dysfunction in AD brain and their cytotoxic mechanisms have been proposed to involve interactions with cell membranes. In this review, we discuss our solid-state nuclear magnetic resonance (ssNMR) studies of Aβ interactions with model membranes.
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ItemO-aryl and Carbonyl Carbon Contents of Food Waste and Biosolid Predict P Availability in an Acidic Soil(Frontiers Media SA, 2021)Organic waste streams, otherwise known as organic amendments (OA), contain potentially valuable nutrients which may additionally increase legacy nutrient availability in soil. This is particularly the case for phosphorus (P) where declining reserves of rock phosphate add an extra dimension to their utility. In acidic soils, OA have been reported to increase P availability through the action of O-aryl and carbonyl groups (represent organic acid compounds) by substituting previously fixed, legacy P and forming organometallic complexes to reduce P sorption. This study aimed to investigate if signature P (orthophosphate) and C (O-aryl and carbonyl) content of OA could be used to predict soil P availability, to replace traditional ways of testing OA and also for future prescriptive applications. Food waste and biosolid were the sources of OA in this study, with pyrolysis and composting processes used to create a range of functional groups. Nuclear magnetic resonance (NMR) spectroscopy was utilized to identify forms of C (solid-state 13C NMR) and P compounds (solution-state 31P NMR) in these OA. The O-aryl, carbonyl, and orthophosphate content were higher in pyrolysis and composted materials compared to their feedstock substrate. The effect of OA addition on soil P availability was monitored in a 110-day laboratory incubation study. Results showed an increase in soil P availability (Olsen P) and a decrease in soil P buffering capacity (PBC) after incubation. The increase in soil P availability was not predicted well by the NMR-derived orthophosphate content of OA, which may be due to the overestimation of plant-available orthophosphate content by the solution-state 31P NMR. Furthermore, an additional increase in soil ΔOlsen P (difference between observed and expected) was obtained above the Olsen P added from OA indicating substitution of previously fixed soil P. Both indices of P availability namely ΔOlsen P (r = 0.63–0.83) and ΔPBC (difference between treatment—control) (r = −0.50 to −0.80) showed strong (but opposite) correlations with the ratio of O-aryl to carbonyl C content of OA. It was concluded that the ratio of O-aryl and carbonyl C content of OA could be used to predict the P availability in acidic soil.