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PubMed2000

Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence.

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.

Animals↗
PubMed2000

Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells.

One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones. In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with vectors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter. In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequate combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of interest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells. Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.

Animals↗
PubMed2003

Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets.

An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 microm) produced by a piezoelectric generator system with 10-microm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 x 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A-fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 microg L(-1) reaching a LOD of 0.04 microg L(-1). The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 microg L(-1) in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 ,g L(-1), with a dynamic range between 4 and 149.5 microg L(-1) in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.

Binding, Competitive↗
PubMed1989

Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme.

Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Effects of intrinsic fluorescence and quenching on fluorescence-based screening of natural products.

To evaluate the effects of intrinsic (natural) fluorescence and quenching as confounding variables in fluorescence-based enzyme inhibition assays of natural products, we measured the fluorescence and quenching properties of 25 components of popular herbal products. The analyses were performed under conditions typically employed in drug-drug interaction studies that use c-DNA-derived P450 isoforms and surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin, quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere with these assays. Intrinsic fluorescence had a greater effect on these assays than quenching and for one compound, yangonin, was sufficient to mask inhibition and potentially produce a false negative result. Quenching had less of an effect on these assays, but was significant enough for one compound, quercetin, to mimic "weak" inhibition. Therefore, because intrinsic fluorescence or quenching could render some natural products unsuitable for testing in certain fluorometric assays, it would be prudent to include an evaluation of these properties in experimental protocols.

Apigenin↗
PubMed2002

Laparoscopic fluorescence diagnosis for intraabdominal fluorescence targeting of peritoneal carcinosis experimental studies.

OBJECTIVE: To assess 5-aminolevulinic acid (ALA)-induced protoporphyrin IX accumulation and fluorescence in peritoneal colon carcinoma metastases and its benefits for laparoscopic fluorescence diagnosis. SUMMARY BACKGROUND DATA: Occult, macroscopically nonvisible peritoneal micrometastases can be missed in laparoscopy or open surgery. Laparoscopic fluorescence diagnosis allows detection of these lesions after intraperitoneal lavage with ALA and subsequent fluorescence induction by blue-light excitation. METHODS: A disseminated peritoneal carcinosis was induced by laparoscopic implantation of colon carcinoma cells (CC531) in the peritoneum of 55 WAG/Rij rats. After 12 days of tumor growth the animals were randomized into 11 groups with different photosensitization parameters. Peritoneal lavage was performed either with 1.5% or 3.0% ALA solution, except for one control group. Photosensitization times were 0.5, 1, 2, 4, or 8 hours. Spectrometry was performed using an optical multichannel analyser. ALA and protoporphyrin IX serum levels were measured by high-performance liquid chromatography to determine systemic load. RESULTS: Protoporphyrin IX tumor accumulation and fluorescence peaked 2 to 4 hours after ALA application in both main groups, 1.5% and 3.0% ALA. Tumor detection rate was most effective in the 1.5% ALA group. Compared with conventional white-light laparoscopy alone, blue-light excitation detected 35% additional intraabdominal tumor foci. CONCLUSIONS: Laparoscopic fluorescence diagnosis can increase the sensitivity and specificity of diagnostic staging laparoscopy. It allows determination of the extent of peritoneal carcinosis. Improved preoperative assessment helps to avoid unnecessary laparotomies and radical resections.

Aminolevulinic Acid↗
PubMed1991

Fluorescence study of the conformational properties of myoglobin structure. 1. pH-dependent changes of tryptophanyl fluorescence in intact and chemically modified sperm whale apomyoglobins.

The pH-dependent fluorescence of intact sperm whale apomyoglobin (apo-Mb) containing two tryptophans at positions 7 and 14, and of apo-Mb derivatives modified on Trp7 by 2-hydroxy-5-nitrobenzyl bromide (Koshland reagent) and o-nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N-terminal alpha-NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo-Mb have been resolved within the pH range 2-12.5. The quantum yield of the 'buried' Trp14 (lambda max at 326 nm) is shown to be twofold higher at pH greater than 8.5 than that of the 'exposed' Trp7 (lambda max at 333 nm). At pH 8.5-5.5 the fluorescence of Trp14 diminished approximately twofold due to quenching by the ionized His residue, most probably His119. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5-3-fold in the pH range 5.5-4.3 while the contribution of Trp14 remains constant. The conformational changes at the N-terminal and in the region adjacent to it, as well as in the whole apo-Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N-terminal part of apo-Mb.

Animals↗
PubMed1991

Fluorescence study of the conformational properties of myoglobin structure. 3. pH-dependent changes in porphyrin and tryptophan fluorescence of the complex of sperm whale apomyoglobin with protoporphyrin IX; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure.

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5-10.8 but change significantly at pH less than 5.5 and pH greater than 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH less than 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH greater than 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintenance of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.

Animals↗
PubMed1996

Escherichia coli isocitrate dehydrogenase kinase/phosphatase. Overproduction and kinetics of interaction with its substrates by using intrinsic fluorescence and fluorescent nucleotide analogues.

The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N-terminus. By using a one-step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern-Volmer quenching constant in the presence of acrylamide (Ksv = 2.1 M-1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl-derivatives ADP, ATP, GDP and GTP and adenosine-5'-triphosphoro-1-(5-sulfonic-acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7-2.5 microM for unmodified ADP or ATP and of 2.5-3.7 microM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10-fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315-340 by using limited proteolysis of IDH K/P by endoproteinase Lys-C. Only one main site of cleavage was obtained: the peptide bond K346-E347 which was strongly protected in the presence of ATP.

Adenosine Triphosphatases↗
PubMed2001

Evidence from action and fluorescence spectra that UV-induced violet-blue-green fluorescence enhances leaf photosynthesis.

We assessed the contribution of UV-induced violet-blue-green leaf fluorescence to photosynthesis in Poa annua, Sorghum halepense and Nerium oleander by measuring UV-induced fluorescence spectra (280-380 nm excitation, 400-550 nm emission) from leaf surfaces and determining the monochromatic UV action spectra for leaf photosynthetic O2-evolution. Peak fluorescence emission wavelengths from leaf surfaces ranged from violet (408 nm) to blue (448 nm), while excitation peaks for these maxima ranged from 333 to 344 nm. Action spectra were developed by supplementing monochromatic radiation from 280 to 440 nm, in 20 nm increments, to a visible nonsaturating background of 500 mumol m-2 s-1 photosynthetically active radiation and measuring photosynthetic O2-evolution rates. Photosynthetic rates tended to be higher with the 340 nm supplement than with higher or lower wavelength UV supplements. Comparing photosynthetic rates with the 340 nm supplement to those with the 400 nm supplement, the percentage enhancement in photosynthetic rates at 340 nm ranged from 7.8 to 9.8%. We suspect that 340 nm UV improves photosynthetic rates via fluorescence that provides violet-blue-green photons for photosynthetic energy conversion because (1) the peak excitation wavelength (340 nm) for violet-blue-green fluorescence from leaves was also the most effective UV wavelength at enhancing photosynthetic rates, and (2) the magnitude of photosynthetic enhancements attributable to supplemental 340 nm UV was well correlated (R2 = 0.90) with the apparent intensity of 340 nm UV-induced violet-blue-green fluorescence emission from leaves.

Magnoliopsida↗
PubMed1988

X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

Aequorin↗
PubMed1983

Fluorescence of histones H1. A tyrosinate-like fluorescence emission in Ceratitis capitata H1 at neutral pH values.

We have examined the fluorescent properties of histones H1, and of some peptides derived from them, from calf thymus and from the fruit fly Ceratitis capitata. The fluorescent emission spectrum of folded histone H1 from C. capitata at neutral pH is characterized by a maximum at 303 nm and a shoulder at 340 nm. The overall quantum yields of fluorescence do not increase upon folding, although the fluorescence of the single tyrosyl residue of calf H1 is enhanced when the protein folds. As expected, the excitation maximum of calf H1 is shifted to longer wavelengths when the protein folds and its position does not depend upon the wavelength at which the fluorescence is observed. However, Ceratitis H1 exhibits two excitation maxima. The first corresponds to emission at 303 nm and it is slightly redshifted upon protein folding, whereas the second, which corresponds to emission at 340 nm, is displaced from 280 nm in the denatured protein to approximately 285 nm in the folded histone. This suggests that the two tyrosyl residues of the insect histone behave as independent fluorophores. The shoulder at 340 nm does not appear at pH 2, even when the protein is folded. Titration to neutral pH values results in the appearance of the shoulder, the process being characterized by a pK'a approximately equal to 3.7. The fluorescence spectrum of insect histone has been resolved into the contributions of the individual tyrosyl residues and the results suggest that the emission at 340 nm originates in a tyrosinate that may be formed in the excited state by proton transfer to the carboxylate anion of a glutamyl residue. The results obtained from these experiments have also aided in resolving the near-UV circular dichroism spectrum of insect histone (Barbero, J.L., Franco, L., Montero, F., and Morán, F. (1980) Biochemistry 19, 4080-4087) into the individual contributions of the tyrosyl residues.

Animals↗
PubMed1997

Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes.

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helices required for receptor function.

Amino Acid Sequence↗
PubMed2003

Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses.

The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. Our method will provide a powerful solution to various problems confronting researchers, such as the photobleaching of dyes in two-photon excitation microscopy.

Fluorescent Dyes↗
PubMed2005

High selective determination iron(II) by its enhancement effect on the fluorescence of pyrene-tetramethylpiperidinyl (TEMPO) as a spin fluorescence probe.

A novel fluorescence method determination for iron(II) with a high selectivity and sensitivity has been proposed, based on the enhancement of fluorescence signals resulting from specific redox reaction between synthesized spin fluorescence probe pyrene-tetramethylpiperidinyl (TEMPO) and iron(II). Under the experimental conditions, fluorescent probe displayed a rapid and linear response for iron(II) over the concentration range from 2.4 x 10(-7) to 3.6 x 10(-6) mol/L. The limit of detection was 4.0 x 10(-8) mol/L. The relative standard deviation of six replicate measurements was 1.90% for 3.0 x 10(-7) mol/L iron(II). Because of the specific redox reaction between developed spin fluorescence probe and iron(II), there are few interference by other ions, especially in the presence of relative high concentration iron(III). The method has been successfully applied for iron(II) determinations in two different kinds of real samples. Results determined by the proposed method agree favorably with those determined UV-vis spectrometry method with 1,10-phenanthroline.

Acids↗
PubMed2002

Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.

Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell except for trace amounts in the nuclear membrane and some vesicles. We performed fluorescence correlation spectroscopy measurements and photon-counting histogram analysis in specific domains of live cells. For AK1-EGFP, we observed only one diffusion component in the cytoplasm. For AK1beta-EGFP, we observed two distinct diffusion components on the plasma membrane. One corresponded to the free diffusing protein, whereas the other represented the membrane-bound AK1beta-EGFP. The diffusion rate of AK1-EGFP was slowed by a factor of 1.8 with respect to that of EGFP, which was 50% more than what we would expect for a free diffusing AK1-EGFP. To rule out the possibility of oligomer formation, we performed photon-counting histogram analysis to direct analyze the brightness difference between AK1-EGFP and EGFP. From our analysis, we concluded that cytoplasmic AK1-EGFP is monomeric. fluorescence correlation spectroscopy proved to be a powerful technique for quantitatively studying the mobility of the target protein in live cells. This technology offers advantages in studying protein interactions and function in the cell.

Adenylate Kinase↗
PubMed2003

Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process.

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.

Biophysical Phenomena↗
PubMed1997

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Escherichia coli↗