scholarly journals Direct visualization of superselective colloid-surface binding mediated by multivalent interactions

2021 ◽  
Vol 118 (36) ◽  
pp. e2106036118
Author(s):  
Christine Linne ◽  
Daniele Visco ◽  
Stefano Angioletti-Uberti ◽  
Liedewij Laan ◽  
Daniela J. Kraft
Keyword(s):  
Colloidal Particles ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Model System ◽  
Receptor Density ◽  
Mobile Dna ◽  
Surface Binding ◽  
Receptor Level ◽  
Foerster Resonance Energy Transfer ◽  
Multivalent Interactions

Reliably distinguishing between cells based on minute differences in receptor density is crucial for cell–cell or virus–cell recognition, the initiation of signal transduction, and selective targeting in directed drug delivery. Such sharp differentiation between different surfaces based on their receptor density can only be achieved by multivalent interactions. Several theoretical and experimental works have contributed to our understanding of this “superselectivity.” However, a versatile, controlled experimental model system that allows quantitative measurements on the ligand–receptor level is still missing. Here, we present a multivalent model system based on colloidal particles equipped with surface-mobile DNA linkers that can superselectively target a surface functionalized with the complementary mobile DNA-linkers. Using a combined approach of light microscopy and Foerster resonance energy transfer (FRET), we can directly observe the binding and recruitment of the ligand–receptor pairs in the contact area. We find a nonlinear transition in colloid-surface binding probability with increasing ligand or receptor concentration. In addition, we observe an increased sensitivity with weaker ligand–receptor interactions, and we confirm that the timescale of binding reversibility of individual linkers has a strong influence on superselectivity. These unprecedented insights on the ligand–receptor level provide dynamic information into the multivalent interaction between two fluidic membranes mediated by both mobile receptors and ligands and will enable future work on the role of spatial–temporal ligand–receptor dynamics on colloid-surface binding.

scholarly journals The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons

2014 ◽  
Vol 111 (5) ◽  
pp. 1033-1045 ◽  
Author(s):  
Christopher G. Vecsey ◽  
Nicolás Pírez ◽  
Leslie C. Griffith
Keyword(s):  
Motor Neurons ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Model System ◽  
Network Activity ◽  
Target Cells ◽  
New Model ◽  
Pigment Dispersing Factor ◽  
Molecular Effects

Neuropeptides have widespread effects on behavior, but how these molecules alter the activity of their target cells is poorly understood. We employed a new model system in Drosophila melanogaster to assess the electrophysiological and molecular effects of neuropeptides, recording in situ from larval motor neurons, which transgenically express a receptor of choice. We focused on two neuropeptides, pigment-dispersing factor (PDF) and small neuropeptide F (sNPF), which play important roles in sleep/rhythms and feeding/metabolism. PDF treatment depolarized motor neurons expressing the PDF receptor (PDFR), increasing excitability. sNPF treatment had the opposite effect, hyperpolarizing neurons expressing the sNPF receptor (sNPFR). Live optical imaging using a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor for cyclic AMP (cAMP) showed that PDF induced a large increase in cAMP, whereas sNPF caused a small but significant decrease in cAMP. Coexpression of pertussis toxin or RNAi interference to disrupt the G-protein Gαo blocked the electrophysiological responses to sNPF, showing that sNPFR acts via Gαo signaling. Using a fluorescent sensor for intracellular calcium, we observed that sNPF-induced hyperpolarization blocked spontaneous waves of activity propagating along the ventral nerve cord, demonstrating that the electrical effects of sNPF can cause profound changes in natural network activity in the brain. This new model system provides a platform for mechanistic analysis of how neuropeptides can affect target cells at the electrical and molecular level, allowing for predictions of how they regulate brain circuits that control behaviors such as sleep and feeding.

scholarly journals Binding and Sensing Properties of a Hybrid Naphthalimide–Pyrene Aza-Cyclophane towards Nucleotides in an Aqueous Solution

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 980
Author(s):  
Aleksandr M. Agafontsev ◽  
Aleksandr S. Oshchepkov ◽  
Tatiana A. Shumilova ◽  
Evgeny A. Kataev
Keyword(s):  
Aqueous Solution ◽  
Fluorescence Spectra ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Selective Recognition ◽  
Dispersion Interactions ◽  
Binding Studies ◽  
Turn On ◽  
Hydrogen Bonding Interactions ◽  
Foerster Resonance Energy Transfer

Selective recognition of nucleotides with synthetic receptors is an emerging direction to solve a series of nucleic acid-related challenges in biochemistry. Towards this goal, a new aza-cyclophane with two different dyes, naphthalimide and pyrene, connected through a triamine linker has been synthesized and studied for the ability to bind and detect nucleoside triphosphates in an aqueous solution. The receptor shows Foerster resonance energy transfer (FRET) in fluorescence spectra upon excitation in DMSO, which is diminished dramatically in the presence of water. According to binding studies, the receptor has a preference to bind ATP (adenosine triphosphate) and CTP (cytidine triphosphate) with a “turn-on” fluorescence response. Two separate emission bands of dyes allow one to detect nucleotides in a ratiometric manner in a broad concentration range of 10−5–10−3 M. Spectroscopic measurements and quantum chemical calculations suggest the formation of receptor–nucleotide complexes, which are stabilized by dispersion interactions between a nucleobase and dyes, while hydrogen bonding interactions of nucleobases with the amine linkers are responsible for selectivity.

scholarly journals Probing DNA - transcription factor interactions using single-molecule fluorescence detection in nanofluidic devices

2021 ◽  
Author(s):  
Mattia Fontana ◽  
Ŝarūnė Ivanovaitė ◽  
Simon Lindhoud ◽  
Willy van den Berg ◽  
Dolf Weijers ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fluorescence ◽  
Dna Oligonucleotides ◽  
Complex Interactions ◽  
Donor And Acceptor ◽  
Nanofluidic Devices ◽  
Foerster Resonance Energy Transfer

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, we combine nanofluidic devices and camera-based, single-molecule Foerster resonance energy transfer (smFRET) detection to study the interactions between plant transcription factors of the Auxin response family (ARF) and DNA oligonucleotides that contain target DNA response elements. In particular, we show that the binding of the unlabelled ARF DNA binding domain (ARF-DBD) to donor and acceptor labelled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, our data on fluorescently labelled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artefacts, enables high-throughput measurements and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

scholarly journals Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation

2021 ◽  
Author(s):  
Erik Jonsson ◽  
Zaw Min Htet ◽  
Jared A.M. Bard ◽  
Ken C Dong ◽  
Andreas Martin
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
26S Proteasome ◽  
Ubiquitin Chain ◽  
Substrate Degradation ◽  
Cellular Environment ◽  
Foerster Resonance Energy Transfer ◽  
Proteolytic Chamber

The 26S proteasome is the major ATP-dependent protease in eukaryotic cells, where it catalyzes the degradation of thousands of proteins for general homeostasis and the control of vital processes. It specifically recognizes appropriate substrates through attached ubiquitin chains and uses its ATPase motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Foerster Resonance Energy Transfer (FRET) measurements to provide unprecedented insights into the mechanisms of selective substrate engagement, ATP-dependent degradation, and the regulation of these processes by ubiquitin chains. Our assays revealed the proteasome conformational dynamics and allowed monitoring individual substrates as they progress through the central channel during degradation. We found that rapid transitions between engagement- and processing-competent conformations of the proteasome control substrate access to the ATPase motor. Ubiquitin-chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and facilitate degradation initiation. The global conformational transitions cease upon substrate engagement, and except for apparent motor slips when encountering stably folded domains, the proteasome remains in processing-competent states for substrate translocation and unfolding, which is further accelerated by ubiquitin chains. Our studies revealed the dependence of ATP-dependent substrate degradation on the conformational dynamics of the proteasome and its allosteric regulation by ubiquitin chains, which ensure substrate selectivity and prioritization in a crowded cellular environment.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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