Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research

Szöllősi, János; Damjanovich, Sándor; Mátyus, László

doi:10.1002/(sici)1097-0320(19980815)34:4<159::aid-cyto1>3.0.co;2-b

Cited by 200 publications

(122 citation statements)

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“…2,6 In particular they are widely used in fluorescence microscopy and FRET studies. 1,19 Unfortunately, fluorescein can exist in different species (prototropic forms) with dissimilar photophysical properties, depending on the environmental pH. 20-,22 Fluorescein in aqueous solution can exist as cationic (FH 3 + ), neutral (FH 2 ), monoanionic (FH -), and dianionic (F 2-) species (Scheme 1), the concentrations of which are determined by the pH.…”

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Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

et al. 2009

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Fluorescein is one of most used fluorescent labels for characterising biological systems, such as proteins, and is used in fluorescence microscopy. However, if fluorescein is to be used for quantitative measurements involving proteins, then one must account for the fact that the fluorescence of fluorescein labelled protein can be affected by the presence of intrinsic amino acids residues, such as, tryptophan (Trp). There is a lack of quantitative information to explain in detail the specific processes that are involved and this makes it difficult to evaluate quantitatively the photophysics of fluorescein labelled proteins. To address this we have explored the fluorescence of fluorescein in buffered solutions, in different acid and basic conditions, and at varying concentrations of tryptophan derivatives, using steady-state absorption and fluorescence spectroscopy, combined with fluorescence lifetime measurements. Stern-Volmer analyses show the presence of static and dynamic quenching processes between fluorescein and tryptophan derivatives. Non-fluorescent complexes with low association constants (5.0 -24.1 M -1 ) are observed at all pH values studied. At low pH values, however, an additional static quenching contribution by a sphereof-action (SOA) mechanism was found. The possibility of a proton transfer mechanism being involved in the SOA static quenching, at low pH, is discussed based on the presence of the different fluorescein prototropic species. For the dynamic quenching process, the bimolecular rate constants obtained (2.5-5.3×10 9 M -1 s -1 ) were close to the Debye-Smoluchowski diffusion rate constants. In the encounter controlled reaction mechanism, a photoinduced electron transfer mechanism was applied using the reduction potentials and charges of the fluorophore and quencher, in addition to the ionic strength of the environment. The electron transfer rate constants (2.3-6.7×10 9 s -1 ) and the electronic coupling values (5.7-25.1 cm -1 ) for fluorescein fluorescence quenching by tryptophan derivatives in the encounter complex were then obtained and analysed. This data will be applied to generate a more detailed, quantitative understanding of the photophysics of fluorescein when conjugated to proteins containing the amino acid tryptophan. KEYWORDS:Fluorescein, Tryptophan, fluorescence quenching, electron transfer, bioconjugation.Page 2 of 19 IntroductionWidely applied in fluorescence imaging microscopy, the fluorophore labelled protein can be used to rapidly and easily visualise many different biochemical pathways, which involve protein interactions, protein expression, trafficking, intracellular signalling events, and cellular location. 1,2 Many of the fluorophores used are designed to conjugate with specific amino acid residues or functional groups present in the target biomolecule. In many cases, the fluorophore is simply used as a contrast agent i.e. to show the location of the target biomolecule in a particular environment. However, for quantitative measurements of protein-surface intera...

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Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

et al. 2009

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“…Here, we have assessed the cell surface organization of the B7 ligands using confocal microscopy-based FRET, a phenomenon that occurs due to nonradiative energy transfer from an excited donor to an acceptor molecule (24,25). Because energy transfer depends on the inverse sixth power of the distance between the two fluorophores, this phenomenon typically reports the proximity of two molecules on a scale of 10-100 Å, a distance that correlates with the size of the molecules on the cell surface (26,27).…”

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Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling

Bhatia

Edidin

Almo

et al. 2005

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

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The costimulatory ligands B7-1 and B7-2 are expressed on the surface of antigen-presenting cells and interact with the costimulatory receptors CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on T cells. Although B7-1 and B7-2 are homologous ligands having common receptors, they exhibit distinct biochemical features and roles in immune regulation. Several biochemical and structural studies have indicated differences in the oligomeric state of B7-1 and B7-2. However, the organization of B7 ligands on the cell surface has not been examined. By using photobleaching-based FRET (pbFRET), we demonstrate that B7-1 and B7-2 adopt different oligomeric states on the cell surface. Our study shows that B7-2 exists as a monomer on the cell surface whereas B7-1 exists predominantly as dimers on the cell surface. A series of mutations in B7-1 result in the expression of a predominantly monomeric species on the cell surface and validate the dimer interface proposed by prior crystallographic analysis. The difference in the oligomeric states of B7-1 and B7-2 provides insight into the geometric organization of the costimulatory receptorligand complexes in the immunological synapse and suggests constraints on signal transduction mechanisms involved in T cell activation.oligomerization ͉ photobleaching FRET ͉ T cell costimulation T he engagement of B7-1 and B7-2 with CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) represents the best characterized T cell costimulatory interactions. CTLA-4 and CD28 share a similar molecular architecture, existing as disulfide linked homodimers of Ig variable (IgV) domains (1, 2). CTLA-4 and CD28 bind the B7 molecules with different affinities (3) and direct different T cell functional outcomes. Interaction of CD28 with B7 ligands delivers a positive signal to T cells that promotes proliferation and cytokine secretion and prevents the induction of T cell tolerance (4, 5). In contrast, the interaction of CTLA-4 with B7 ligands attenuates T cell activation and induces T cell anergy (6-8). Furthermore, recent studies have shown that CD28 and CTLA-4 binding to B7 ligands delivers different signals to dendritic cells. Binding of CD28 to B7 ligands leads to up-regulation of IL-6 production by dendritic cells, resulting in immunostimulatory activity (9). On the other hand, binding of CTLA-4 to B7 ligands up-regulates IFN-␥, which, in turn, up-regulates the expression of the enzyme indolamine, 2,3-dioxygenase in dendritic cells, resulting in tryptophan catabolism and suppression of T cell proliferation (10, 11).B7-1 and B7-2 are both type 1 transmembrane proteins with a membrane distal IgV and a membrane proximal Ig constant (IgC) domain and share Ϸ25% sequence identity. Although interacting with the same receptors, B7-1 and B7-2 show several distinct features (12, 13). B7-1 binds CTLA-4 and CD28 with equilibrium dissociation constants (K d ) of 0.2 M and 4 M, respectively, whereas B7-2 exhibits Ϸ5-to 10-fold lower affinities, with K d values of 2.6 M and 20 M for CTLA-4 a...

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“…The higher Stokes shift is accomplished by means of fluorescence resonance energy transfer (FRET) between the donor molecule (e.g., phycoerythrin) and acceptor molecule (e.g., cyanine 7) (2-5). The optimum energy transfer level depends on the acceptor-to-donor molar ratio, distance, and orientation and results in significant reduction of donorderived emission, while self-quenching of the acceptor is avoided (3,6,7). The delicate balance between donor and acceptor is the reason that emission spectra of tandem dyes are affected by manufacturing and protein conjugation processes as well as by an apparent instability of fluorescence characteristics.…”

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“…The instability (i.e., degradation) of tandem dyes is caused by photon-induced oxidation and results in relative short reagent shelf-lives. Despite the widely accepted notion that tandem dyes demand protection from light exposure to minimize changes in fluorescence characteristics (4,6,(8)(9)(10)(11)(12), essentially no literature is available that describes these changes quantitatively.…”

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Flow cytometry and the stability of phycoerythrin‐tandem dye conjugates

Hulspas¹,

Dombkowski

Preffer

et al. 2009

Cytometry Pt A

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Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies. ' 2009 International Society for Advancement of CytometryKey terms degradation; fluorochrome; stability; T-regulatory cells; CD4/CD8; clinical; flow cytometry; compensation THE emission spectra of most fluorochromes are fairly constant. The conjugation of fluorochromes to proteins (e.g., antibodies) may affect their quantum efficiency (i.e., absolute fluorescence intensity) but shifts their entire spectrum by only a few nanometers (1). In contrast, tandem dyes are known to display a considerable degree of variability in fluorescence intensities within different regions of each emission spectrum. A tandem dye is a combination of two fluorochromes (donor and acceptor dye) such that the difference between the excitation and emission maxima (Stokes shift) is significantly increased relative to that of the donor dye. The higher Stokes shift is accomplished by means of fluorescence resonance energy transfer (FRET) between the donor molecule (e.g., phycoerythrin) and acceptor molecule (e.g., cyanine 7) (2-5). The optimum energy transfer level depends on the acceptor-to-donor molar ratio, distance, and orientation and results in significant reduction of donorderived emission, while self-quenching of the acceptor is avoided (3,6,7). The delicate balance between donor and acceptor is the reason that emission spectra of tandem dyes are affected by manufactur...

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Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and research

Cited by 200 publications

References 122 publications

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling

Flow cytometry and the stability of phycoerythrin‐tandem dye conjugates

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