Fluorescence correlation spectroscopy as a probe of molecular dynamics

Aragón, Sergio R.; Pecora, R.

doi:10.1063/1.432357

Cited by 329 publications

(278 citation statements)

References 15 publications

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“…15 is governed by the following equation of motion (11,25) dp(β, Ω, t|α, Ω 0 , 0)/dt = − Here, L i is an angular momentum operator and R is the rate matrix for transitions, as before. We use the diagonal form of the diffusion operator, L i D β ij L j , because the body-fixed frame diagonalizes this operator.…”

Section: Methodsmentioning

confidence: 99%

“…First, we decompose the motion of n into global reorientation of a body-fixed reference frame (BF) coordinate system, and the trajectory of the vector in the body-fixed frame, n BF . Then, the total correlation function can be decomposed into contributions from each of the species in the exchange-diffusion system (11,25), each with its own BF coordinate system. Eq.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Influence of the coupling of interdomain and overall motions on NMR relaxation

Wong

Case²,

Szabó³

2009

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

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Most theoretical models for NMR relaxation in liquids assume that overall rotational motion can be described as rotational diffusion with a single diffusion tensor. Such models cannot handle motions (such as between "closed" and "open" states of an enzyme, or between conformers of a partially disordered system) where the shape of the molecule (and hence its rotational diffusion behavior) fluctuates. We provide here a formalism for dealing with such problems. The model involves jumps between discrete conformers with different overall diffusion tensors, and a master (rate) equation to describe the transitions between these conformers. Numerical examples are given for a two-site jump model where global and local motions are concerted, showing how the rate of conformational transitions (relative to the rate of rotational diffusion) affects the observed relaxation parameters.rotation | diffusion | spin T he interpretation of NMR relaxation parameters in terms of molecular parameters has a long and successful history. The "classic" analysis, which is widely used, describes rotational tumbling by a diffusion equation with a single (perhaps anisotropic) diffusion tensor; superimposed on this are internal motions (of various amplitudes and timescales) that are assumed to be independent of overall rotation (1). The internal motions themselves may be modeled as diffusive motion, such as diffusion in a cone or in a more general restraining potential (2, 3), or may be treated as jumps between a set of discrete conformers, where populations and rate constants of conversion among these species are specified in a master equation (4-6).In many interesting cases, however, the assumption that rotational tumbling can be described with a single diffusion tensor is not realistic. These include large-amplitude interdomain motions and proteins that are at least partially disordered. In both cases, there are significant populations of conformations with quite different shapes and hence diffusion tensors. The timescale of such global conformational changes can be similar to that of the overall reorientational dynamics, further complicating the problem.At present, approaches used to analyze NMR relaxation in such systems effectively assume that the overall tumbling and interdomain motions are uncorrelated. The simplest such procedure is to use the extended model-free approach (7), as has been done by Baber et al. for calmodulin (8), where it is assumed that the overall tumbling motion can be described by the diffusion tensor of the dynamically averaged structure and the slow internal motion is identified with the interdomain motions with respect to this frame. More recently, Ryabov and Fushman introduced the ITS (interconversion between two states) model and applied it to di-ubiquitin (9). With respect to overall and interdomain motions, their model is simply a two-site jump model (6, 10) with full anisotropic (but independent) overall motion.In this article we consider the simplest class of models where internal and overall tumbling moti...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Influence of the coupling of interdomain and overall motions on NMR relaxation

Wong

Case²,

Szabó³

2009

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

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“…Time traces of photons were recorded with a single photon avalanche photodiode (PDM-50 mm, PicoQuant) for green fluorescence detection and a commercial data acquisition system (HydraHarp 400, PicoQuant) with a picosecond time resolution. The diffusion time t d can be extracted from the fit of the correlated data using the following equation 52,53 :…”

Section: Molecular Biology and Protein Expression/purificationmentioning

confidence: 99%

Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

2014

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In living organisms, most proteins work in complexes to form multicomponent protein machines. The function of such multicomponent machines is usually addressed by dividing them into a collection of two state systems at equilibrium. Many molecular machines, like Hsp90, work far from equilibrium by utilizing the energy of ATP hydrolysis. In these cases, important information is gained from the observation of the succession of more than two states in a row. We developed a four-colour single-molecule FRET system to observe the succession of states in the heat shock protein 90 (Hsp90) system, consisting of an Hsp90 dimer, the cochaperone p23 and nucleotides. We show that this multicomponent system is a directional ATP-dependent machinery. This reveals a previously undescribed mechanism on how cochaperones can modify Hsp90, namely by strengthening of the coupling between ATP hydrolysis and a kinetic step involved in the Hsp90 system resulting in a stronger directionality.

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“…FCS has been used to study a wide variety of problems ranging from hydrodynamics to reaction kinetics (Aragon and Pecora, 1976;Magde et al, 1974). Previously, both FCS, and time-resolved fluorescence anisotropy of long lifetime probes have been used to study the rotational diffusion of small molecules in solution (Kask et al, 1987(Kask et al, , 1988Mets 2001).…”

Section: Introductionmentioning

confidence: 99%

Polarized fluorescence correlation spectroscopy of DNA‐DAPI complexes

Barcellona

Gammon

Hazlett

et al. 2004

Microscopy Res & Technique

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We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

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Fluorescence correlation spectroscopy as a probe of molecular dynamics

Cited by 329 publications

References 15 publications

Influence of the coupling of interdomain and overall motions on NMR relaxation

Influence of the coupling of interdomain and overall motions on NMR relaxation

Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

Polarized fluorescence correlation spectroscopy of DNA‐DAPI complexes

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