Fluorescence correlation spectroscopy

Ries, Jonas; Schwille, Petra

doi:10.1002/bies.201100111

Cited by 220 publications

(171 citation statements)

References 59 publications

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“…2A). To extract characteristic time scales of fluctuations, we calculated the fluorescence intensity autocorrelation function < I(t)·I(t+τ)>, where I denotes fluorescence intensity, t is time, and τ is the time delay (27)(28)(29). For all conditions, we observed a double-exponential decay, indicating the presence of two different time scales (Fig.…”

Section: Resultsmentioning

confidence: 97%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Richardson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

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Section: Resultsmentioning

confidence: 97%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Richardson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…A laser excites fluorescent molecules in a diffraction-limited and pinholeconfined volume within a tissue. Fluorescence fluctuations are correlated over time and compared with transport model predictions (Ries and Schwille, 2012). FCS thus allows analysis of local transport processes over very short time and length scales.…”

Section: Evidence For Hindered Diffusion Of Nodalmentioning

confidence: 99%

Morphogen transport

et al. 2013

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The graded distribution of morphogens underlies many of the tissue patterns that form during development. How morphogens disperse from a localized source and how gradients in the target tissue form has been under debate for decades. Recent imaging studies and biophysical measurements have provided evidence for various morphogen transport models ranging from passive mechanisms, such as free or hindered extracellular diffusion, to cell-based dispersal by transcytosis or cytonemes. Here, we analyze these transport models using the morphogens Nodal, fibroblast growth factor and Decapentaplegic as case studies. We propose that most of the available data support the idea that morphogen gradients form by diffusion that is hindered by tortuosity and binding to extracellular molecules.

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“…Autocorrelation analysis has found numerous applications in fluorescence correlation spectroscopy, although it has not been used for the kinetic analysis of equilibrium fluctuations in single-molecule force spectroscopy (36,37). Autocorrelation signals for time traces recorded with and without HP35WT at 8 pN are shown in Fig.…”

Section: Significancementioning

confidence: 99%

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Žoldák

Stigler

Pelz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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In this study we expand the accessible dynamic range of singlemolecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 μs thus approaching the timescales accessible for all atom molecular dynamics simulations. P rotein folding is a spontaneous process where a linear polypeptide chain acquires a functional and highly complex 3D structure. Conformational folding can occur on timescales ranging from hours down to microseconds (1, 2). The study of small fast-folding proteins has provided key information for understanding fundamental aspects of protein-folding mechanisms. Recently, it has even become possible to simulate folding trajectories of small proteins in full atomic detail using molecular dynamics simulations up to the millisecond time range (3, 4). However, direct time-resolved experimental measurements of such ultrafast processes have largely been limited to a few ensemble methods like temperature-jump, continuous-flow experiments, and triplet-lifetime measurements.The C-terminal subdomain of the actin-binding protein villin (villin headpiece, HP35) has been used as a model system in a number of both experimental and simulation studies of protein folding at the speed limit (5-19). The folding kinetics of HP35 has been studied extensively mostly by T-jump and by tripletlifetime experiments (6-8, 11, 12, 16). Folding of the wild-type HP35 at 30°C occurs at 348 × 10 3 s −1 , and unfolding at 7.4 × 10 3 s −1 , which in combination yields a free energy of folding of ∼4 k B T (6). Before crossing the major folding barrier, HP35 undergoes a rapid local rearrangement on the nanosecond timescale, which results in stable contacts of tertiary structure. Triplettriplet-energy transfer (TTET) measurements have reported a native-state heterogeneity and an additional high-energy intermediate on the native-state side of the major unfolding barrier (16). A detailed structural picture of the locking-unlocking reaction of the native states as observed in TTET data were given by recent all-atom molecular dynamics simulations (5). Along this line, a variety of native conformations was found consiste...

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Fluorescence correlation spectroscopy

Cited by 220 publications

References 59 publications

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Morphogen transport

Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

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