scholarly journals Click-Coupling to Electrostatically Grafted Polymers Greatly Improves the Stability of a Continuous Monitoring Sensor with Single-Molecule Resolution

ACS Sensors ◽  
2021 ◽  
Author(s):  
Yu-Ting Lin ◽  
Rosan Vermaas ◽  
Junhong Yan ◽  
Arthur M. de Jong ◽  
Menno W.J. Prins
Keyword(s):  
Single Molecule ◽  
Continuous Monitoring ◽  
Grafted Polymers ◽  
The Stability ◽  
Click Coupling

scholarly journals Energetic dependencies dictate folding mechanism in a complex protein

2019 ◽  
Vol 116 (51) ◽  
pp. 25641-25648 ◽  
Author(s):  
Kaixian Liu ◽  
Xiuqi Chen ◽  
Christian M. Kaiser
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Large Scale ◽  
Elongation Factor ◽  
Multidomain Protein ◽  
Domain Interactions ◽  
The Stability ◽  
Domain Iii ◽  
And Function ◽  
Terminal Domains

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Assembly and Dynamics of the Bacterial Flagellum

2020 ◽  
Vol 74 (1) ◽  
pp. 181-200 ◽  
Author(s):  
Judith P. Armitage ◽  
Richard M. Berry
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Complex Structure ◽  
Live Cells ◽  
Flagellar Motor ◽  
Interacting Protein ◽  
Bacterial Flagellum ◽  
Bacterial Flagellar Motor ◽  
The Stability

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45–50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

Bilateral Surveillance Guidance Note

Policy Papers ◽  
2010 ◽  
Vol 2010 (112) ◽  
Author(s):  
Keyword(s):  
Continuous Monitoring ◽  
International Monetary System ◽  
Executive Board ◽  
Financial Policies ◽  
Monetary System ◽  
Guidance Note ◽  
Review Mechanism ◽  
The Stability ◽  
Policy Adjustments ◽  
Core Activity

This note provides guidance to staff on the conduct of bilateral surveillance, a core activity of the Fund. Surveillance involves the continuous monitoring of members’ economic and financial policies, and regular Article IV consultations. During these consultations, staff holds pointed discussions with country authorities on the economic situation, the authorities’ policies, and desirable policy adjustments. These discussions are then reported to the Fund’s Executive Board for its consideration. The goal is, through thorough analysis, candid discussions, and a peer-review mechanism, to promote the domestic and external stability of members’ economies and thereby the stability of the international monetary system as a whole.

scholarly journals Site-Specific One-to-One Click Coupling of Single Proteins to Individual Carbon Nanotubes: A Single-Molecule Approach

2017 ◽  
Vol 139 (49) ◽  
pp. 17834-17840 ◽  
Author(s):  
Mark Freeley ◽  
Harley L. Worthy ◽  
Rochelle Ahmed ◽  
Ben Bowen ◽  
Daniel Watkins ◽  
...  
Keyword(s):  
Carbon Nanotubes ◽  
Single Molecule ◽  
Site Specific ◽  
One To One ◽  
Click Coupling

scholarly journals Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Junyi Jiao ◽  
Mengze He ◽  
Sarah A Port ◽  
Richard W Baker ◽  
Yonggang Xu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Sm Proteins ◽  
Single Molecule Force Spectroscopy ◽  
Helix Bundle ◽  
Four Helix Bundle ◽  
The Stability ◽  
Sm Protein ◽  
Template Complex

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

scholarly journals The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

2019 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Md Simulations ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Single Molecule Fret ◽  
Allosteric Mechanism ◽  
Specific Transport ◽  
Slc6 Family ◽  
The Stability

AbstractNeurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

scholarly journals Probing the stability of the SpCas9-DNA complex after cleavage

2021 ◽  
Author(s):  
Pierre Aldag ◽  
Fabian Welzel ◽  
Leonhard Jakob ◽  
Andreas Schmidbauer ◽  
Marius Rutkauskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Slowing Down ◽  
Target Strand ◽  
Dna Strands ◽  
Dna Strand ◽  
The Stability ◽  
The Individual ◽  
Dna Complex

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target.

scholarly journals Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anne E Dodson ◽  
Jasper Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Single Molecule ◽  
Genome Stability ◽  
Gene Repression ◽  
Cell Divisions ◽  
Rna Fish ◽  
Eukaryotic Gene ◽  
The Stability ◽  
Lines Of Evidence

Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability. However, to date there has been no quantitative evaluation of the stability of heterochromatic gene repression. We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent, heritable changes in genotype and phenotype. This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions. In concordance with multiple lines of evidence suggesting these events were rare and transient, single-molecule RNA FISH showed that transcription was limited. The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1, a silencing establishment factor, in the maintenance and/or inheritance of silencing. In addition, we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state. These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible.

scholarly journals Microcephaly family protein MCPH1 stabilizes RAD51 filaments

2020 ◽  
Vol 48 (16) ◽  
pp. 9135-9146
Author(s):  
Hao-Yen Chang ◽  
Chia-Yi Lee ◽  
Chih-Hao Lu ◽  
Wei Lee ◽  
Han-Lin Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Fold Increase ◽  
Functional Study ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Mammalian Expression ◽  
Contact Points ◽  
Family Protein ◽  
The Stability

Abstract Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2–RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51–ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

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