scholarly journals Diffusivity Measurement by Single-Molecule Recycling in a Capillary Microchannel

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 800
Author(s):  
Bo Wang ◽  
Lloyd M. Davis
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
High Throughput Screening ◽  
Fused Silica ◽  
Bulk Solution ◽  
Confined Space ◽  
Binding Studies ◽  
Observation Window ◽  
Laser Focus ◽  
Inner Surface

Microfluidic devices have been extensively investigated in recent years in fields including ligand-binding analysis, chromatographic separation, molecular dynamics, and DNA sequencing. To prolong the observation of a single molecule in aqueous buffer, the solution in a sub-micron scale channel is driven by a electric field and reversed after a fixed delay following each passage, so that the molecule passes back and forth through the laser focus and the time before irreversible photobleaching is extended. However, this practice requires complex chemical treatment to the inner surface of the channel to prevent unexpected sticking to the surface and the confined space renders features, such as a higher viscosity and lower dielectric constant, which slow the Brownian motion of the molecule compared to the bulk solution. Additionally, electron beam lithography used for the fabrication of the nanochannel substantially increases the cost, and the sub-micron dimensions make the molecule difficult to locate. In this paper, we propose a method of single-molecule recycling in a capillary microchannel. A commercial fused-silica capillary with an inner diameter of 2 microns is chopped into a 1-inch piece and is fixed onto a cover slip. Two o-rings on the sides used as reservoirs and an o-ring in the middle used as observation window are glued over the capillary. The inner surface of the capillary is chemically processed to reduce the non-specific sticking and to improve capillary effect. The device does not require high-precision fabrication and thus is less costly and easier to prepare than the nanochannel. 40 nm Fluospheres® in 50% methanol are used as working solution. The capillary is translated by a piezo stage to recycle the molecule, which diffuses freely through the capillary, and a confocal microscope is used for fluorescence collection. The passing times of the molecule through the laser focus are calculated by a real-time control system based on an FPGA, and the commands of translation are given to the piezo stage through a feedback algorithm. The larger dimensions of the capillary overcomes the strong sticking, the reduced diffusivity, and the difficulty of localizing the molecule. We have achieved a maximum number of recycles of more than 200 and developed a maximum-likelihood estimation of the diffusivity of the molecule, which attains results of the same magnitude as the previous report. This technique simplifies the overall procedure of the single-molecule recycling and could be useful for the ligand-binding studies in high-throughput screening.

scholarly journals High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography

IUCrJ ◽  
2019 ◽  
Vol 6 (6) ◽  
pp. 1074-1085 ◽  
Author(s):  
Tadeo Moreno-Chicano ◽  
Ali Ebrahim ◽  
Danny Axford ◽  
Martin V. Appleby ◽  
John H. Beale ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Radiation Damage ◽  
High Throughput ◽  
High Throughput Screening ◽  
Room Temperature ◽  
Data Sets ◽  
Binding Studies ◽  
X Ray ◽  
Temperature Damage ◽  
Serial Femtosecond Crystallography

High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.

scholarly journals Interbase-FRET binding assay for pre-microRNAs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mattias Bood ◽  
Anna Wypijewska del Nogal ◽  
Jesper R. Nilsson ◽  
Fredrik Edfeldt ◽  
Anders Dahlén ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Drug Targets ◽  
Structural Changes ◽  
Small Sample Size ◽  
Resonance Energy ◽  
Small Sample ◽  
Titration Calorimetry ◽  
Drug Candidate ◽  
Binding Studies ◽  
Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

scholarly journals Back protection of canvas paintings

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Tim Padfield ◽  
Nicolas Padfield ◽  
Daniel Sang-Hoon Lee ◽  
Anne Thøgersen ◽  
Astrid Valbjørn Nielsen ◽  
...  
Keyword(s):  
Fluid Dynamics ◽  
Computational Fluid Dynamics ◽  
Relative Humidity ◽  
Temperature Variation ◽  
Room Temperature ◽  
Single Point ◽  
Confined Space ◽  
Constant Wall Temperature ◽  
Back Plate ◽  
Inner Surface

Abstract In this paper different scenarios for back protection of a canvas painting and their effect on the stability of the relative humidity behind the painting are tested. A painting on canvas, stretched on a wooden frame, was fitted with various styles of back protection and then exposed to a cycle of temperature variation at the back, with the front exposed to a constant room temperature. The painting was also exposed to a constant wall temperature and varying room temperature. The space between the canvas and the back board was fitted with temperature and relative humidity (RH) sensors. The sensors were used to provide the essential single-point data of temperature and RH at the given locations. For more comprehensive understanding of the rather confined space, further numerical simulation (computational fluid dynamics) was adopted as part of the investigation. The computational fluid dynamics was used to understand the natural convection within the microclimate through the depictions of temperature distribution, as well as the corresponding airflow. The unprotected painting suffered a large RH variation at its back, because of the varying canvas temperature interacting with the constant room air moisture content. Effective stabilisation of the RH behind the canvas against temperature variation was provided by a shiny aluminium alloy sheet sealed against the frame. The non-absorbent back board experienced a strong variation in RH, because of humidity buffering of the space by the painting canvas at a different temperature. Either a space or insulation between this back plate and the wall reduced the risk of condensation on the inner surface of the back plate. Insulation will however increase the risk of condensation on the wall surface behind the painting. An absorbent back board de-stabilised the RH at the painting canvas surface by providing a competing humidity buffer at a different temperature. To provide protection against moisture exchange with an unsuitable room RH, extra humidity buffer was placed 3 mm behind the painting canvas, kept close to the painting temperature by insulation between this buffer and the back board. This stabilised RH at the canvas surface but increased both the temperature and the RH variation at the back board and thus increased the risk of condensation on the inner surface of the back board. The RH and the temperature in the narrow spaces between the painting canvas and the wooden stretcher frame were always more nearly constant than in the open canvas area, which suggests an explanation for the widely observed better condition of the areas of canvas paintings which lie close over the support structure. Our conclusion is that a non-absorbent, impermeable back plate gives good RH stability against a changing temperature gradient between wall and canvas painting surface.

The identification of cytoplasmic membrane calcium-entry channels by ligand-binding studies

1984 ◽  
Vol 12 (6) ◽  
pp. 941-943 ◽  
Author(s):  
H. GLOSSMANN ◽  
A. GOLL ◽  
D. R. FERRY ◽  
M. ROMBUSCH
Keyword(s):  
Ligand Binding ◽  
Cytoplasmic Membrane ◽  
Calcium Entry ◽  
Binding Studies

scholarly journals Comparative studies on peptides representing the so-called tachykinin-like region of the Alzheimer Aβ peptide [Aβ(25–35)]

1998 ◽  
Vol 336 (2) ◽  
pp. 419-427 ◽  
Author(s):  
Omar M. A. EL-AGNAF ◽  
G. Brent IRVINE ◽  
Geraldine FITZPATRICK ◽  
W. Kenneth GLASS ◽  
David J. S. GUTHRIE
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Comparative Studies ◽  
Aβ Peptide ◽  
Amyloid Protein ◽  
Binding Studies ◽  
Nk1 Receptors ◽  
Ligand Displacement ◽  
Β Amyloid

In an attempt to answer the question of whether or not the so-called tachykinin-like region of the Alzheimer β-amyloid protein [Aβ(25–35)] can act as a tachykinin, the sequences Aβ(25–35), Aβ(25–35)amide and their norleucine-35 and phenylalanine-31 analogues were synthesized. These peptides were examined with ligand binding studies, electron microscopy, CD and NMR. In all cases some differences were found between the Aβ(25–35) analogue and the corresponding Phe31 peptide. In addition, in ligand displacement studies on tachykinin NK1 receptors, only the Phe31 analogue showed activity comparable to that of genuine tachykinins. We conclude that peptides based on Aβ(25–35) but with a Phe residue at position 31 do display properties typical of a tachykinin, but that peptides with Ile at this position do not.

scholarly journals Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor

2018 ◽  
Vol 115 (46) ◽  
pp. 11688-11693 ◽  
Author(s):  
Thomas Suren ◽  
Daniel Rutz ◽  
Patrick Mößmer ◽  
Ulrich Merkel ◽  
Johannes Buchner ◽  
...  
Keyword(s):  
Free Energy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Mechanical Load ◽  
Force Spectroscopy ◽  
Folding Pathway ◽  
Hormone Binding ◽  
Single Molecule Force Spectroscopy

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a “lid” structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of 41 kBT (24 kcal/mol). The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional 12 kBT of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s−1. From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

scholarly journals Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

2021 ◽  
Author(s):  
Endang Purba ◽  
Ei-ichiro Saita ◽  
Reetesh Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Electron Tomography ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Cancer Therapies ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

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