scholarly journals Abrogation of prenucleation, transient oligomerization of the Huntingtin exon 1 protein by human profilin I

2020 ◽  
Vol 117 (11) ◽  
pp. 5844-5852 ◽  
Author(s):  
Alberto Ceccon ◽  
Vitali Tugarinov ◽  
Rodolfo Ghirlando ◽  
G. Marius Clore
Keyword(s):  
Single Molecule ◽  
Chemical Exchange ◽  
Coiled Coil ◽  
Relaxation Dispersion ◽  
Line Broadening ◽  
Exon 1 ◽  
Nmr Techniques ◽  
Mechanistic Basis ◽  
Kinetics Of ◽  
Micromolar Range

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington’s disease. Here, we investigate the interaction of profilin with httex1using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex≤ 50 to 70 μs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex∼750 μs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (Kdiss∼ 17 and ∼ 31 μM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τex∼ 600 μs andKdiss∼ 50 μM). Finally, we demonstrate that, in stable profilin–httex1complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1tetramers, is completely abolished, and only the pathway resulting in “nonproductive” dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.

Notizen: Binding and Kinetics of 1-Methylimidazol on Fe(III)-Protoporphyrin(IX)dimethylester and Fe(III)-Tetraphenylporphyrin in Chloroform

1976 ◽  
Vol 31 (2) ◽  
pp. 242-245 ◽  
Author(s):  
Eberhard V. Goldammer ◽  
Herbert Zorn
Keyword(s):  
Spin State ◽  
Chemical Exchange ◽  
Phase 1 ◽  
Protoporphyrin Ix ◽  
Bulk Phase ◽  
Paramagnetic Species ◽  
Solvent Exchange ◽  
Line Broadening ◽  
Axial Ligands ◽  
Kinetics Of

Nuclear magnetic line widths data have been used to determine the rate of solvent exchange between the coordination sphere of Fe(III)-protoporphyrin(IX)dimethylester or Fe(III)-tetraphenylporphyrin and the bulk phase of 1-methylimidazol/chloroform. At temperatures below 322 K both porphyrins are in the low-spin state and separate PMR absorption caused by the methyl protons of two 1-methylimidazol molecules complexed in fifth and sixth position of ferri-porphyrins is detected. At T ≳ 320 K an accelerated exchange of these ligands was observed and the underlying kinetic parameters have been extracted. It was found that this exchange takes place when the paramagnetic species is in its low-spin state. For 240 K ≲ T ≲ 290 K dynamic line broadening of bulk phase 1-methylimidazol indicates occurrence of chemical exchange attributed to 1-methylimidazol interacting with ferri-porphyrin in addition to the strongly bound axial ligands.

scholarly journals Single molecule mechanics of the kinesin neck

2009 ◽  
Vol 106 (17) ◽  
pp. 6992-6997 ◽  
Author(s):  
Thomas Bornschlögl ◽  
Günther Woehlke ◽  
Matthias Rief
Keyword(s):  
Single Molecule ◽  
Leucine Zipper ◽  
Structural Integrity ◽  
Mechanical Stability ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Force Microscopy ◽  
Atomic Force ◽  
Kinetics Of ◽  
High Mechanical Stability

Structural integrity as well as mechanical stability of the parts of a molecular motor are crucial for its function. In this study, we used high-resolution force spectroscopy by atomic force microscopy to investigate the force-dependent opening kinetics of the neck coiled coil of Kinesin-1 from Drosophila melanogaster. We find that even though the overall thermodynamic stability of the neck is low, the average opening force of the coiled coil is >11 pN when stretched with pulling velocities >150 nm/s. These high unzipping forces ensure structural integrity during motor motion. The high mechanical stability is achieved through a very narrow N-terminal unfolding barrier if compared with a conventional leucine zipper. The experimentally mapped mechanical unzipping profile allows direct assignment of distinct mechanical stabilities to the different coiled-coil subunits. The coiled-coil sequence seems to be tuned in an optimal way to ensure both mechanical stability as well as motor regulation through charged residues.

scholarly journals The condensin complex is a mechanochemical motor that translocates along DNA

2017 ◽  
Author(s):  
Tsuyoshi Terekawa ◽  
Shveta Bisht ◽  
Jorine M. Eeftens ◽  
Cees Dekker ◽  
Christian H. Haering ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Average Distance ◽  
Coiled Coil ◽  
Chromosome Organization ◽  
Single Molecule Imaging ◽  
Base Pairs ◽  
Translocation Mechanism ◽  
Mechanistic Basis

One Sentence SummarySingle-molecule imaging reveals that eukaryotic condensin is a highly processive DNA-translocating motor complex.AbstractCondensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensin’s translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ∼60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ∼50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.

Full-length-transcriptomic analysis in mice and human heart using Single-Molecule Real-time Sequencing (SMRT) identified 15 novel isoforms and a novel promoter region of PGC1-alpha

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
D Oehler ◽  
A Goedecke ◽  
A Spychala ◽  
K Lu ◽  
N Gerdes ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Molecule ◽  
High Fat Diet ◽  
Human Heart ◽  
Full Length ◽  
Funding Source ◽  
Smrt Sequencing ◽  
High Fat ◽  
Exon 1 ◽  
Novel Exon

Abstract Background Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R). Methods RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR. Results Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter. In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet. Conclusion Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Single-Molecule Force-Clamp Experiments Reveal Kinetics of Mechanically Activated Silyl Ester Hydrolysis

ACS Nano ◽  
2012 ◽  
Vol 6 (2) ◽  
pp. 1314-1321 ◽  
Author(s):  
Sebastian W. Schmidt ◽  
Pavel Filippov ◽  
Alfred Kersch ◽  
Martin K. Beyer ◽  
Hauke Clausen-Schaumann
Keyword(s):  
Single Molecule ◽  
Ester Hydrolysis ◽  
Silyl Ester ◽  
Force Clamp ◽  
Kinetics Of

scholarly journals Increasing the time resolution of single-molecule experiments with Bayesian inference

2017 ◽  
Author(s):  
Colin D. Kinz-Thompson ◽  
Ruben L. Gonzalez
Keyword(s):  
Bayesian Inference ◽  
Single Molecule ◽  
Time Resolution ◽  
Rate Constants ◽  
Computational Approach ◽  
Biomolecular Systems ◽  
Time Resolved ◽  
The Given ◽  
Kinetics Of ◽  
Resolution Data

AbstractMany time-resolved, single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian Inference for the Analysis of Sub-temporal-resolution Data (BIASD) approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.

scholarly journals Biochemistry: one molecule at a time

2021 ◽  
Vol 65 (1) ◽  
pp. 1-3
Author(s):  
Dominika T. Gruszka
Keyword(s):  
Complex Networks ◽  
Single Molecule ◽  
Population Level ◽  
Early Career ◽  
Molecular Heterogeneity ◽  
Biological Processes ◽  
Biological Macromolecule ◽  
Exciting Field ◽  
Early Career Researchers ◽  
Mechanistic Basis

Abstract Biological processes are orchestrated by complex networks of molecules. Conventional approaches for studying the action of biomolecules operate on a population level, averaging out any inhomogeneities within the ensemble. Investigating one biological macromolecule at a time allows researchers to directly probe individual behaviours, and thus characterise the intrinsic molecular heterogeneity of the system. Single-molecule methods have unravelled unexpected modes of action for many seemingly well-characterised biomolecules and often proved instrumental in understanding the intricate mechanistic basis of biological processes. This collection of reviews aims to showcase how single-molecule techniques can be used to address important biological questions and to inspire biochemists to ‘zoom in’ to the population and probe individual molecular behaviours, beyond the ensemble average. Furthermore, this issue of Essays in Biochemistry is the very first written and edited entirely by early career researchers, and so it also highlights the strength, diversity and excellence of the younger generation single-molecule scientists who drive this exciting field of research forward.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

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