Fluorescence Lifetime Standards for Time and Frequency Domain Fluorescence Spectroscopy

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca 21 concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca 21 /Mg 21 association to DREAM. Our data indicate a minor impact of Ca 21 association on the overall structure of the Nand C-terminal domains, although Ca 21 binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca 21 bound forms of the protein. Time-resolved fluorescence data indicate that Ca 21 binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca 21 association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca 21 and Mg 21 suggesting that, under physiological conditions, Ca 21 free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

show abstract

“…The reference compound used in lifetime measurements was 2,5-diphenyloxazole (PPO) in ethanol (s 5 1.40 ns). 45 …”

Section: Time-resolved Fluorescence Measurementsmentioning

confidence: 99%

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The temporal resolution of time-and frequency-domain instrumentation can be tested by means of fluorescence lifetime standards, as detailed elsewhere [57]. In addition, the alignment of the excitation and emission polarizers can be checked with a very dilute scattering solution, as in a steady-state instrument.…”

Section: Iv4 Checking the Accuracy Of Time-resolved Anisotropy Measumentioning

confidence: 99%

“…As the statistical properties of the data are known, one can use these criteria to evaluate the fit of a model to the experimental data as described for total intensity decays (e.g., Boens et al [57]). …”

mentioning

confidence: 99%

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

Ameloot

vandeVen

Acuña

et al. 2013

Pure and Applied Chemistry

Self Cite

View full text Add to dashboard Cite

After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measure ments are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.

show abstract

“…All the fluorescence intensity measurements were carried out on non-deaerated samples at room temperature. 25 Quenching experiments: Because of solubility issues in buffered solutions, at pH 5.0, 7.4, and 11.0, stock solutions of 0.1 M AcTrp in 0.2 M NaOH were first prepared. Solutions of AcTrp at pH 5.0, 7.4, and 11.0 were generated by carefully monitoring, using a pH meter, the addition of small volumes of concentrated HCl.…”

Section: Fluorescence Quantum Yieldsmentioning

confidence: 99%

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

et al. 2009

View full text Add to dashboard Cite

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...

show abstract

Fluorescence Lifetime Standards for Time and Frequency Domain Fluorescence Spectroscopy

Cited by 400 publications

References 67 publications

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

Contact Info

Product

Resources

About

Fluorescence Lifetime Standards for Time and Frequency Domain Fluorescence Spectroscopy

Cited by 400 publications

References 67 publications

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

Contact Info

Product

Resources

About

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator