Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules

Ehrenberg, Måns; Rigler, Rudolf

doi:10.1017/s003358350000216x

Cited by 94 publications

(42 citation statements)

References 14 publications

(16 reference statements)

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“…Quantitative measurements of membrane dynamics are possible with fluorescence recovery after photobleaching (13,14), single-particle tracking techniques (8,15,16), and optical trapping by laser tweezers (13,17,18). Fluorescence correlation spectroscopy, a method with tradition in the study of reaction kinetics and molecular interactions in solution (19,20), also has been applied to the study of cellular systems recently (21,22). The method allows the determination of absolute molecular concentration, mobility, and comobility in small, confocal volume elements of living cells (23).…”

Section: Advanced Cell Biophysical and Molecular Biological Methodolomentioning

confidence: 99%

Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model

Vereb

Szöllősi

Matkó

et al. 2003

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

472

385

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The fluid mosaic membrane model proved to be a very useful hypothesis in explaining many, but certainly not all, phenomena taking place in biological membranes. New experimental data show that the compartmentalization of membrane components can be as important for effective signal transduction as is the fluidity of the membrane. In this work, we pay tribute to the Singer-Nicolson model, which is near its 30th anniversary, honoring its basic features, ''mosaicism'' and ''diffusion,'' which predict the interspersion of proteins and lipids and their ability to undergo dynamic rearrangement via Brownian motion. At the same time, modifications based on quantitative data are proposed, highlighting the often genetically predestined, yet flexible, multilevel structure implementing a vast complexity of cellular functions. This new ''dynamically structured mosaic model'' bears the following characteristics: emphasis is shifted from fluidity to mosaicism, which, in our interpretation, means nonrandom codistribution patterns of specific kinds of membrane proteins forming smallscale clusters at the molecular level and large-scale clusters (groups of clusters, islands) at the submicrometer level. The cohesive forces, which maintain these assemblies as principal elements of the membranes, originate from within a microdomain structure, where lipid-lipid, protein-protein, and protein-lipid interactions, as well as sub-and supramembrane (cytoskeletal, extracellular matrix, other cell) effectors, many of them genetically predestined, play equally important roles. The concept of fluidity in the original model now is interpreted as permissiveness of the architecture to continuous, dynamic restructuring of the molecular-and higherlevel clusters according to the needs of the cell and as evoked by the environment.

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Section: Advanced Cell Biophysical and Molecular Biological Methodolomentioning

confidence: 99%

Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model

Vereb

Szöllősi

Matkó

et al. 2003

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

472

385

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“…It is thus ideally suited for studying cellular dynamics on the molecular level. The quantitative analysis of such single molecule experiments is readily performed by studying the fluctuations of the fluorescence signal using well established techniques such as fluorescence correlation spectroscopy (FCS) [10,11]. However, these techniques are also diffraction-limited, and standard confocal microscopy usually averages the details of nanoscale molecular dynamics.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)

Leutenegger¹,

Ringemann²,

Lasser³

et al. 2012

Opt. Express

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We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40-50 nm was attained which was ultimately limited by the low lateral signal-tobackground ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.

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“…On the other hand, evolutionary approaches make use of rapid and highly sensitive screening assays to detect minute quantities of better adapted individuals among a large excess of alternatives. For all these purposes, fluorescence correlation spectroscopy (FCS) (5)(6)(7)(8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled biomolecules that undergo temporal changes in their diffusion properties (10)(11)(12)(13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

Kettling

Koltermann

Schwille

et al. 1998

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

291

224

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A method for sensitively monitoring enzyme kinetics and activities by using dual-color f luorescence crosscorrelation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two f luorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and f luorophores at each 5 end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K M of 14 ؎ 1 nM and a k cat of 4.6 ؎ 0.2 min ؊1 . In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color f luorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.Kinetic studies on enzymes are among the most important tools for understanding biological interactions at the molecular level. In combination with new approaches in genetic engineering and structure determination, there have been major efforts in recent years to develop more sensitive and precise techniques for characterizing the kinetics of enzyme reactions. These techniques have made it possible to develop efficient assays for analyzing catalytic parameters such as turnover rates, substrate specificity, as well as regio-and stereospecificity; they constitute a major part of the biochemical and pharmaceutical research. Moreover, the alteration of catalytic properties, either by evolutionary biotechnology (1-4) or by rational approaches, is of special interest for medical and industrial applications. The desired features are as follows: accelerated reaction rates; higher, relaxed, or even new substrate specificities; tolerance to nonnatural environments; and novel types of reactions. Modern rational design is the interplay between computer modeling and experimental testing of designed molecules by sensitive and quantitative assays. On the other hand, evolutionary approaches make use of rapid and highly sensitive screening assays to detect minute quantities of better adapted individuals among a large excess of alternatives. For all these purposes, fluorescence correlation spectroscopy (FCS) (5-8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled ...

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Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules

Cited by 94 publications

References 14 publications

Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model

Dynamic, yet structured: The cell membrane three decades after the Singer–Nicolson model

Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)

Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy

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