Photophysical properties of Cerulean and Venus fluorescent proteins

Sarkar, Priyabrata; Koushik, Srinagesh V.; Vogel, Steven S.; Gryczyński, Ignacy; Gryczyński, Zygmunt

doi:10.1117/1.3156842

Cited by 46 publications

(46 citation statements)

References 34 publications

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“…The fluorescence decay of eGFP and mVenus could be approximated well with a single exponential (Fig. 5, A and B, and Table 2) and the resulting lifetimes agreed well with reported values (46,47). The fluorescence decay of mCherry in the F RR (t) PIE channel could be fitted reasonably well with a single exponential model and the recovered lifetime was 1.50 5 0.06 ns, close to what has been reported as the major component governing the fluorescence decay of mCherry (25).…”

Section: Resolving Fret Species With Lifetime-weighted Pie-fisupporting

confidence: 79%

Pulsed Interleaved Excitation Fluctuation Imaging

Hendrix

Schrimpf

Höller

et al. 2013

Biophysical Journal

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Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.

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Section: Resolving Fret Species With Lifetime-weighted Pie-fisupporting

confidence: 79%

Pulsed Interleaved Excitation Fluctuation Imaging

Hendrix

Schrimpf

Höller

et al. 2013

Biophysical Journal

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“…7A). When a high molecular weight fluorophore, such as Venus, is excited, the orientation of emitted light is typically highly correlated with the orientation of the electric field of the light used for excitation, resulting in a high anisotropy value (32). This arises primarily because large fluorophores, such as GFP and its derivatives, have slow rotational motion relative to their fluorescence lifetime.…”

Section: Resultsmentioning

confidence: 99%

A Single Amino Acid Change in Inhibitory Killer Cell Ig-like Receptor Results in Constitutive Receptor Self-Association and Phosphorylation

Kumar

Sarkar

Sim

et al. 2015

The Journal of Immunology

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Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within immunoreceptor tyrosine-based inhibition motifs (ITIM) results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work has shown that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. Here we show that substitution of histidine 36 (His-36) in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, and recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine (KIR2DL1-H36F), maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases, and that KIR binding to HLA-C must override the hindrance His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C+ target cells than expression of wild type KIR2DL1. These results have revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.

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“…Fluorescence of the tryptophan residue in HSA provides information on the local environment of the protein. In fact, tryptophan residues in proteins are largely used to study dynamics and structure of proteins by measuring fluorescence intensity and anisotropy decays [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%