Triple-Color Coincidence Analysis: One Step Further in Following Higher Order Molecular Complex Formation

Heinze, Katrin G.; Jahnz, Michael; Schwille, Petra

doi:10.1016/s0006-3495(04)74129-6

Cited by 86 publications

(77 citation statements)

References 29 publications

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“…Performing FCS on three channels would identify populations of intermediates that contain S7, S9, and S19. However, attempts to date have been based upon coincidence methods (23,24) that are not rigorously compatible with FCS and FCCS and cannot distinguish double and triple-labeled particles at high concentrations.…”

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confidence: 99%

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Ridgeway

Millar²,

Williamson

2012

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

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The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.fluorescence cross-correlation spectroscopy | fluorescence correlation spectroscopy | RNA-protein interactions T he complete assembly mechanism for the 30S ribosome has remained elusive. Approximate rate constants and orders of binding are known from kinetic experiments, but kinetic profiles for individual protein binding and RNA folding steps are complex and defy simple models (1-8). The fundamentally kinetic process of ribosome assembly is thus still best understood in terms of the thermodynamic (9) maps generated by the Nomura group in the 1970s (10). The Nomura maps documented how the omission of one protein precludes incorporation of others, but only monitored stable protein-RNA interactions that would survive the biochemical purification steps including nonequilibrium ultracentrifugation. Recent RNA protection data suggest that weak protein-RNA interactions form rapidly during assembly (5, 6), raising the question of how completely the map represents the types of intermediates that form during assembly.Nomura dependencies exist between three specific proteins, S7, S9, and S19, that bind the 16S rRNA (Fig. 1A) late in assembly (2, 4-8). In principle, binding of these three proteins to RNA could occur by any and all possible parallel pathways that are il...

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confidence: 99%

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Ridgeway

Millar²,

Williamson

2012

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

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“…5 The concentrations of individual species labeled with spectrally distinct fluorescent tags and the kinetics of any subsequent complex formation have been successfully resolved with FCCS using either one or two excitation sources. [6][7][8] Due to the stringent alignment requirements in combining two excitation sources, the use of two-photon excitation overcame these technical disadvantages and facilitated the excitation of multicolor fluorescent mixtures. 9,10 For example, two-photon excitation of up to three dyes has been implemented to simultaneously resolve molecular concentrations and kinetics of three distinctly labeled, interacting species.…”

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confidence: 99%

“…9,10 For example, two-photon excitation of up to three dyes has been implemented to simultaneously resolve molecular concentrations and kinetics of three distinctly labeled, interacting species. 8,11 More recently, one-photon excitation has been implemented for FCCS studies, whereby a single laser line has been used to excite two fluorophores with similar excitation but different emission characteristics. 12 Complexes formed from the reactants labeled with different color fluorophores results in a new spectrally distinct species with a correlation trace that can be fit to quantify the kinetics, dynamics, and concentration of this complex.…”

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confidence: 99%

“…12 Complexes formed from the reactants labeled with different color fluorophores results in a new spectrally distinct species with a correlation trace that can be fit to quantify the kinetics, dynamics, and concentration of this complex. 6,9,13 Although the cross-correlation signal is specific to doubly labeled (or triply labeled) species, the accurate recovery of the dynamics and concentrations of the complex is critically dependent on the emission spectra of the labels. 5,14 For fluorescent labels that do not have cleanly separated spectra, the reliability of multicolor FCCS methodologies becomes limited as a result of spectral cross talk.…”

mentioning

confidence: 99%

“…[6][7][8]16 In addition, dichroics and barrier filters are carefully chosen to maximize signal and minimize cross talk to alleviate the degradation of FCCS signals. 5 Thus, there exists a lower limit for detecting reaction complexes based on multicolor cross-correlation analysis and other analysis approaches such as photon counting histogram and moment analysis.…”

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confidence: 99%

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Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

et al. 2008

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Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto-and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.Fluorescence correlation spectroscopy (FCS) was developed to study the temporal correlations of thermodynamic concentration fluctuations in a reactive chemical system and was described in a series of papers by Magde, Webb, and Elson. 1,2 FCS monitors temporal variations of the fluorescence signal in an optically defined focal volume. These intensity fluctuations originate from important dynamic properties, such as diffusion, aggregation, chemical and enzyme kinetics, flow, and active transport. [1][2][3] The amplitude of these fluctuations is proportional to the square root of the number of molecules in the volume. 3,4

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Design of Modern Biopharmaceuticals by Ultra‐high‐throughput Screening and Directed Evolution

Rarbach¹,

Coco²,

Koltermann³

et al. 2005

Modern Biopharmaceuticals

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Triple-Color Coincidence Analysis: One Step Further in Following Higher Order Molecular Complex Formation

Cited by 86 publications

References 29 publications

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

Spectrally Resolved Fluorescence Correlation Spectroscopy Based on Global Analysis

Design of Modern Biopharmaceuticals by Ultra‐high‐throughput Screening and Directed Evolution

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