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

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.

scholarly journals Dark noise and retinal degeneration from D190N-rhodopsin

2020 ◽  
Vol 117 (37) ◽  
pp. 23033-23043 ◽  
Author(s):  
Daniel Silverman ◽  
Zuying Chai ◽  
Wendy W. S. Yue ◽  
Sravani Keerthi Ramisetty ◽  
Sowmya Bekshe Lokappa ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Outer Segment ◽  
Segment Length ◽  
Single Molecule Level ◽  
Dark Noise ◽  
Order Of Magnitude ◽  
Human Mutation ◽  
Unclear Etiology

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.

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

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.

scholarly journals Coexistence of an ILPR i-Motif and a Partially Folded Structure with Comparable Mechanical Stability Revealed at the Single-Molecule Level

2010 ◽  
Vol 132 (26) ◽  
pp. 8991-8997 ◽  
Author(s):  
Soma Dhakal ◽  
Joseph D. Schonhoft ◽  
Deepak Koirala ◽  
Zhongbo Yu ◽  
Soumitra Basu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Stability ◽  
Single Molecule Level ◽  
Folded Structure

scholarly journals Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

2019 ◽  
Author(s):  
Antoine Vigouroux ◽  
Baptiste Cordier ◽  
Andrey Aristov ◽  
Enno Oldewurtel ◽  
Gizem Özbaykal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Single Molecule ◽  
Cell Shape ◽  
Mechanical Stability ◽  
Cell Envelope ◽  
Cylindrical Part ◽  
Single Molecule Level ◽  
Class A ◽  
Wall Stiffness ◽  
Peptidoglycan Cell Wall

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

Atomic Force Microscopy (AFM) for Topography and Recognition Imaging at Single Molecule Level

2013 ◽  
pp. 102-112
Author(s):  
Memed Duman ◽  
Andreas Ebner ◽  
Christian Rankl ◽  
Jilin Tang ◽  
Lilia A. Chtcheglova ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Recognition Imaging

Curaxin-Induced DNA Topology Alterations Trigger the Distinct Binding Response of CTCF and FACT at the Single-Molecule Level

Biochemistry ◽  
2021 ◽  
Vol 60 (7) ◽  
pp. 494-499
Author(s):  
Ke Lu ◽  
Cuifang Liu ◽  
Yinuo Liu ◽  
Anfeng Luo ◽  
Jun Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Topology ◽  
Single Molecule Level
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