scholarly journals Human telomere repeat binding factor TRF1 replaces TRF2 bound to shelterin core hub TIN2 when TPP1 is absent

2018 ◽  
Author(s):  
Tomáš Janovič ◽  
Martin Stojaspal ◽  
Pavel Veverka ◽  
Denisa Horáková ◽  
Ctirad Hofr
Keyword(s):  
Complex Formation ◽  
Single Molecule ◽  
Binding Capacity ◽  
Molecular Model ◽  
Correlation Spectroscopy ◽  
End Joining ◽  
Telomere Repeat ◽  
Single Molecule Level ◽  
Binding Factor ◽  
Confocal Volume

AbstractHuman telomeric repeat binding factors TRF1, TRF2 along with TIN2 form a core of shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence Cross-Correlation Spectroscopy (FCCS) was used to describe the complex formation by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substituted TRF2 on TIN2. We assessed the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces allosteric changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.

Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy

2002 ◽  
Vol 31 (3) ◽  
pp. 241-241 ◽  
Author(s):  
Lutz Jermutus ◽  
Reto Kolly ◽  
Zeno Földes-Papp ◽  
Jozef Hanes ◽  
Rudolf Rigler ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Fluorescence Correlation

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

scholarly journals Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy

2019 ◽  
Vol 116 (23) ◽  
pp. 11247-11252 ◽  
Author(s):  
Toru Kondo ◽  
Jesse B. Gordon ◽  
Alberta Pinnola ◽  
Luca Dall’Osto ◽  
Roberto Bassi ◽  
...  
Keyword(s):  
Correlation Function ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Light Harvesting ◽  
Building Blocks ◽  
Complex Stress ◽  
Correlation Spectroscopy ◽  
Light Harvesting Complex ◽  
Single Molecule Level

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

Characterization of Pteridines: a New Approach by Fluorescence Correlation Spectroscopy and Analysis of Assay Sensitivity

Pteridines ◽  
2001 ◽  
Vol 12 (4) ◽  
pp. 147-153 ◽  
Author(s):  
U. Demel ◽  
Z. Foldes-Papp ◽  
D. Fuchs ◽  
G. P. Tilz
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Cell Mediated Immunity ◽  
Distribution Analysis ◽  
Assay Sensitivity ◽  
New Approach ◽  
Single Molecule Level ◽  
Fluorescence Correlation

Abstract In the present investigation, fluorescence con-elation spectroscopy (FCS) was used to measure the molecular motion of the pteridine derivative neopterin. However, technical limitations in the present optical setup precluded the identification of ,single neopterin molecules. FCS measurements with a fluorophore were also can-ied out for comparison. Exemplified by rhodamine green, we have introduced a concept that allows the detection, identification and analysis of assays in solution at the single-molecule level in tenns of bulk concentration. This concept is based on FCS and Poisson distribution analysis of assay sensitivity. The molecules had not to be quantified in a more concentrated fonn, or in flow and trapping experiments. The study demonstrated an ultrasensitive, reliable, rapid and direct tool for analytics and diagnostics in solution. We discuss a possible application of our new concept in activation control of cell-mediated immunity via neopterin determination.

scholarly journals FRET at the Single Molecule Level using Molecular Brightness and Fluorescence Correlation Spectroscopy

2019 ◽  
Vol 116 (3) ◽  
pp. 567a ◽  
Author(s):  
Robert C. Miller ◽  
Rowan Simonet ◽  
Christin Libal ◽  
Cody Aplin ◽  
Anh Cong ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Fluorescence Correlation ◽  
Molecular Brightness

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level

2017 ◽  
Vol 8 (23) ◽  
pp. 5785-5791 ◽  
Author(s):  
Xuzhu Zhang ◽  
Evangelos Sisamakis ◽  
Krzysztof Sozanski ◽  
Robert Holyst
Keyword(s):  
Complex Formation ◽  
Single Molecule ◽  
Rate Constants ◽  
Single Molecule Level

scholarly journals A fluorescence correlation spectrometer for measurements in cuvettes

2018 ◽  
Author(s):  
B Sahoo ◽  
TB Sil ◽  
B Karmakar ◽  
K Garai
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Diffusion Time ◽  
Correlation Spectroscopy ◽  
Ease Of Use ◽  
Fluorescence Correlation ◽  
Wide Range ◽  
Correlation Spectrometer ◽  
Confocal Volume ◽  
Working Distance

ABSTRACTWe have developed a fluorescence correlation spectroscopy (FCS) setup for performing single molecule measurements on samples inside regular cuvettes. We built this by using an Extra Long Working Distance (ELWD), 0.7 NA, air objective with working distance > 1.8 mm. We have achieved counts per molecule > 44 kHz, diffusion time < 64 μs for rhodamine B in aqueous buffer and a confocal volume < 2 fl. The cuvette-FCS can be used for measurements over a wide range of temperature that is beyond the range permitted in the microscope-based FCS. Finally, we demonstrate that cuvette-FCS can be coupled to automatic titrators to study urea dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette-FCS in the experiments that are regularly performed in fluorimeters but are generally avoided in microscope-based FCS.

Theory of Measuring the Selfsame Single Fluorescent Molecule in Solution Suited for Studying Individual Molecular Interactions by SPSM-FCS

Pteridines ◽  
2002 ◽  
Vol 13 (3) ◽  
pp. 73-82 ◽  
Author(s):  
Zeno Földes-Papp
Keyword(s):  
Single Molecule ◽  
Molecular Interactions ◽  
Spatial Separation ◽  
Absolute Number ◽  
Theoretical Concept ◽  
Correlation Spectroscopy ◽  
Single Individual ◽  
Fluorescent Molecule ◽  
Single Molecule Level

Abstract Three exact criteria are first derived for the probabilities that determine single molecule sensitivity in single-phases, for example in solutions or membranes. It is shown how the criteria can be used to decide whether single molecule sensitivity is obtained. Most straightforwardly, all we require is that we experimentally determine the Poisson probability for the absolute number of fluorescent molecules in the volume of observation. This is achieved by fluorescence correlation spectroscopy and allows identifying the selfsame, one single fluorescent molecule in single-phases. Further, we first provide the new and powerful analytical capability to fluorescencetagged inidividual molecules and reactions in in-vitro and living systems to follow their molecular interactions and function by means of spatial stochastic behavior. This fully stochastic modeling is derived which describes the influence of spatial separation on the reaction coordinate of one single individual molecule in single-phases within the observation volume. The theoretical concept is applied to experimental data 'at the single-molecule level' of photosensitive riboflavin and riboflavin-containing blue fluorescence protein. The data were taken from the literature. As simulation results of the elucidative examples, experiments are suggested that would measure individual, single molecules of flavins and flavoproteins m solution. They are of importance for experimentalists.

scholarly journals BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

2014 ◽  
Vol 207 (5) ◽  
pp. 599-613 ◽  
Author(s):  
Marcel Reuter ◽  
Alex Zelensky ◽  
Ihor Smal ◽  
Erik Meijering ◽  
Wiggert A. van Cappellen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Physical Interaction ◽  
Single Molecule Level ◽  
Before And After ◽  
Endogenous Loci

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.

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