Dark noise and retinal degeneration from D190N-rhodopsin

Numerous rhodopsin mutations have been implicated in night blindness and retinal degeneration, often with unclear etiology. D190N-rhodopsin (D190N-Rho) is a well-known inherited human mutation causing retinitis pigmentosa. Both higher-than-normal spontaneous-isomerization activity and misfolding/mistargeting of the mutant protein have been proposed as causes of the disease, but neither explanation has been thoroughly examined. We replaced wild-type rhodopsin (WT-Rho) in RhoD190N/WT mouse rods with a largely “functionally silenced” rhodopsin mutant to isolate electrical responses triggered by D190N-Rho activity, and found that D190N-Rho at the single-molecule level indeed isomerizes more frequently than WT-Rho by over an order of magnitude. Importantly, however, this higher molecular dark activity does not translate into an overall higher cellular dark noise, owing to diminished D190N-Rho content in the rod outer segment. Separately, we found that much of the degeneration and shortened outer-segment length of RhoD190N/WT mouse rods was not averted by ablating rod transducin in phototransduction—also consistent with D190N-Rho’s higher isomerization activity not being the primary cause of disease. Instead, the low pigment content, shortened outer-segment length, and a moderate unfolded protein response implicate protein misfolding as the major pathogenic problem. Finally, D190N-Rho also provided some insight into the mechanism of spontaneous pigment excitation.

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Single molecule vs. large area design of molecular electronic devices incorporating an efficient 2-aminepyridine double anchoring group

Nanoscale ◽

10.1039/c9nr05662a ◽

2019 ◽

Vol 11 (34) ◽

pp. 15871-15880 ◽

Cited By ~ 4

Author(s):

L. Herrer ◽

A. Ismael ◽

S. Martín ◽

D. C. Milan ◽

J. L. Serrano ◽

...

Keyword(s):

Electrical Properties ◽

Single Molecule ◽

Electronic Devices ◽

Large Area ◽

Molecular Electronic ◽

Single Molecule Level ◽

Order Of Magnitude ◽

Anchoring Group ◽

Molecular Skeleton ◽

Molecular Electronic Devices

The electrical properties of a bidentate molecule in both large area devices and at the single molecule level have been explored and exhibit a conductance one order of magnitude higher than that of monodentate materials with same molecular skeleton.

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Infrared Nanospectroscopy Reveals the Molecular Interaction Fingerprint of an Aggregation Inhibitor with Single Aβ42 Oligomers

10.1101/2020.06.24.168997 ◽

2020 ◽

Author(s):

Francesco Simone Ruggeri ◽

Johnny Habchi ◽

Sean Chia ◽

Michele Vendruscolo ◽

Tuomas P. J. Knowles

Keyword(s):

Small Molecules ◽

Single Molecule ◽

Protein Misfolding ◽

Molecular Mechanisms ◽

Chemical Sensitivity ◽

Single Molecule Level ◽

Incomplete Knowledge ◽

Parkinson’S Diseases ◽

Aggregation Inhibitor ◽

Misfolding Diseases

ABSTRACTVery significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule level morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, originally an anticancer drug capable recently shown to be able to inhibit Aβ42 aggregation in animal models of Alzheimer’s disease. Our results demonstrate that the carbonyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as powerful tool in structure-based drug discovery for protein misfolding diseases.

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Single-molecule level control of host-guest interactions in metallocycle-C60 complexes

Nature Communications ◽

10.1038/s41467-019-12534-6 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Jian-Hong Tang ◽

Yueqi Li ◽

Qingqing Wu ◽

Zixiao Wang ◽

Songjun Hou ◽

...

Keyword(s):

Single Molecule ◽

Guest Molecule ◽

Scanning Tunneling ◽

Level Control ◽

Tunneling Microscopy ◽

Central Importance ◽

Single Molecule Level ◽

Order Of Magnitude ◽

Different Shapes ◽

Mechanical Stretching

Abstract Host−guest interactions are of central importance in many biological and chemical processes. However, the investigation of the formation and decomplexation of host−guest systems at the single-molecule level has been a challenging task. Here we show that the single-molecule conductance of organoplatinum(II) metallocycle hosts can be enhanced by an order of magnitude by the incorporation of a C60 guest molecule. Mechanically stretching the metallocycle-C60 junction with a scanning tunneling microscopy break junction technique causes the release of the C60 guest from the metallocycle, and consequently the conductance switches back to the free-host level. Metallocycle hosts with different shapes and cavity sizes show different degrees of flexibility to accommodate the C60 guest in response to mechanical stretching. DFT calculations provide further insights into the electronic structures and charge transport properties of the molecular junctions based on metallocycles and the metallocycle-C60 complexes.

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Impact of Small Molecules on Intermolecular G-Quadruplex Formation

Molecules ◽

10.3390/molecules24081570 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1570 ◽

Cited By ~ 2

Author(s):

Prabesh Gyawali ◽

Keshav GC ◽

Yue Ma ◽

Sanjaya Abeysirigunawardena ◽

Kazuo Nagasawa ◽

...

Keyword(s):

Small Molecules ◽

Single Molecule ◽

The Other ◽

Single Molecule Level ◽

G Quadruplex ◽

Dna Strands ◽

Order Of Magnitude ◽

Single Molecule Studies ◽

Quadruplex Formation ◽

The Impact

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

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Polypeptide-Solvent Interactions Measured by Single Molecule Force Spectroscopy

MRS Proceedings ◽

10.1557/proc-0898-l06-06-nn04-06 ◽

2005 ◽

Vol 898 ◽

Author(s):

Alexei Valiaev ◽

Dong Woo Lim ◽

Ashutosh Chilkoti ◽

Scott Schmidler ◽

Stefan Zauscher

Keyword(s):

Single Molecule ◽

Chemical Properties ◽

Force Spectroscopy ◽

Hydrophobic Hydration ◽

Peptide Sequence ◽

Segment Length ◽

Single Molecule Force Spectroscopy ◽

Single Molecule Level ◽

Solvent Quality ◽

Stimulus Responsive

AbstractStimulus-responsive biomolecules have attracted a large research interest because of their potential application in various areas such as drug delivery, actuators and sensing devices at the nanoscale. Using single-molecule force spectroscopy (SMFS) we studied elastin-like polypeptides (ELPs). These stimulus-responsive polypeptides undergo an inverse temperature transition, accompanied by a large conformational change, when the solvent quality is changed by increasing the temperature or by addition of salt. Understanding the relationship between peptide sequence and mechanisms of force generation can provide a route to engineer ELPs with desirable mechano-chemical properties. Here we studied the effect of solvent quality and type of guest residue on the mechanical properties of ELPs on the single-molecule level. We used a statistical approach to estimate polymer elasticity parameters from model fits to the data. With this approach we were able to resolve small changes in the Kuhn segment length distributions associated with different molecular architectures. We then show that these mechanical differences likely arise from differences in the hydrophobic hydration of sidegoups, in line with recent predictions from molecular dynamics simulations.

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Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

International Journal of Molecular Sciences ◽

10.3390/ijms22010055 ◽

2020 ◽

Vol 22 (1) ◽

pp. 55

Author(s):

Yue Ding ◽

Dimitra Apostolidou ◽

Piotr Marszalek

Keyword(s):

Single Molecule ◽

Protein Misfolding ◽

Mechanical Stability ◽

Firefly Luciferase ◽

Renilla Luciferase ◽

Protein Activity ◽

Single Molecule Level ◽

Single Domain Protein ◽

Reporter Assays ◽

Bioluminescent Protein

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

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A nanopore interface for high bandwidth DNA computing

10.1101/2021.12.30.474555 ◽

2021 ◽

Author(s):

Karen Zhang ◽

Yuan-Jyue Chen ◽

Kathryn Doroschak ◽

Karin Strauss ◽

Luis Ceze ◽

...

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Dna Computing ◽

Direct Detection ◽

New Paradigm ◽

Single Molecule Level ◽

Oxford Nanopore ◽

Dna Strand Displacement ◽

Order Of Magnitude ◽

Dna Strand

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this method's utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

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Creating conjugated hydrogel encapsulated membranes

MRS Proceedings ◽

10.1557/proc-0950-d09-01 ◽

2006 ◽

Vol 950 ◽

Author(s):

Tae-Joon Jeon ◽

Noah Malmstadt ◽

Jacob Schmidt

Keyword(s):

Ion Channel ◽

Lipid Bilayer ◽

Single Molecule ◽

Lipid Bilayer Membranes ◽

Single Molecule Level ◽

Channel Proteins ◽

Head Groups ◽

Order Of Magnitude ◽

Previous State ◽

Ion Channel Proteins

ABSTRACTDevice engineering for ion channel proteins requires developing systems that incorporate mechanically stable, long-lived lipid bilayer membranes. Building on our previous work, we have further increased lipid bilayer longevity through covalent conjugation of lipid molecules in the bilayer to an encapsulating hydrogel. This is accomplished by polymerizing the hydrogel in situ around a gigaohm-seal membrane containing vinyl-modified lipid head groups, forming a conjugated hydrogel encapsulated membrane (cgHEM). Membranes formed in this manner show remarkable stability, maintaining gigaohm-level resistance for over 270 hours, better than an order-of-magnitude improvement over the previous state of the art. They also demonstrate the capacity to support the incorporation and measurement of ion channel proteins at the single-molecule level.

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