scholarly journals Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

2019 ◽  
Vol 20 (17) ◽  
pp. 4086 ◽  
Author(s):  
Penzkofer ◽  
Silapetere ◽  
Hegemann
Keyword(s):  
Schiff Base ◽  
Melting Temperature ◽  
Absorption Maximum ◽  
Voltage Sensor ◽  
Quantum Yields ◽  
Excitation Spectra ◽  
Thermal Dynamics ◽  
Absorption And Emission ◽  
Absorption Increase ◽  
Long Time

QuasAr1 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense by directed evolution. Here we report absorption and emission spectroscopic studies of QuasAr1 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of QuasAr1 was studied by long-time attenuation coefficient measurements at room temperature (23 ± 2 °C) and at 2.5 ± 0.5 °C. The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 65 ± 3 °C). In the protein melting process the originally present protonated retinal Schiff base (PRSB) with absorption maximum at 580 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Long-time storage of QuasAr1 at temperatures around 2.5 °C and around 23 °C caused gradual protonated retinal Schiff base isomer changes to other isomer conformations, de-protonation to retinal Schiff base isomers, and apoprotein structure changes showing up in ultraviolet absorption increase. Reaction coordinate schemes are presented for the thermal protonated retinal Schiff base isomerizations and deprotonations in parallel with the dynamic apoprotein restructurings.

scholarly journals Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2

2020 ◽  
Vol 21 (18) ◽  
pp. 6576
Author(s):  
Alfons Penzkofer ◽  
Arita Silapetere ◽  
Peter Hegemann
Keyword(s):  
Schiff Base ◽  
Melting Temperature ◽  
Absorption Maximum ◽  
Room Temperature ◽  
Voltage Sensor ◽  
Quantum Yields ◽  
Excitation Spectra ◽  
Thermal Dynamics ◽  
Absorption And Emission ◽  
Refrigerator Temperature

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

scholarly journals Photocycle Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1

2019 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
Alfons Penzkofer ◽  
Arita Silapetere ◽  
Peter Hegemann
Keyword(s):  
Schiff Base ◽  
Quantum Yield ◽  
Absorption Band ◽  
Absorption Spectra ◽  
Absorption Maximum ◽  
Light Exposure ◽  
Voltage Sensor ◽  
Proton Release ◽  
Protonation State ◽  
State Changes

The retinal photocycle dynamics of the fluorescent voltage sensor QuasAr1 (Archaerhodopsin 3 P60S-T80S-D95H-D106H-F161V mutant from Halorubrum sodomense) in pH 8 Tris buffer was studied. The samples were photoexcited to the first absorption band of the protonated retinal Schiff base (PRSB) Ret_580 (absorption maximum at λmax ≈ 580 nm), and the retinal Schiff base photoisomerization and protonation state changes were followed by absorption spectra recordings during light exposure and after light exposure. Ret_580 turned out to be composed of two protonated retinal Schiff base isomers, namely Ret_580I and Ret_580II. Photoexcitation of Ret_580I resulted in barrier-involved isomerization to Ret_540 (quantum yield ≈ 0.056) and subsequent retinal proton release leading to Ret_410 deprotonated retinal Schiff base (RSB). In the dark, Ret_410 partially recovered to Ret_580I and partially stabilized to irreversible Ret_400 due to apoprotein restructuring (Ret_410 lifetime ≈ 2 h). Photoexcitation of Ret_580II resulted in barrier-involved isomerization to Ret_640 (quantum yield ≈ 0.00135) and subsequent deprotonation to Ret_370 (RSB). In the dark, Ret_370 partially recovered to Ret_580II and partially stabilized to irreversible Ret_350 due to apoprotein restructuring (Ret_370 lifetime ≈ 10 h). Photocycle schemes and reaction coordinate diagrams for Ret_580I and Ret_580II were developed and photocyle parameters were determined.

The Steady-State and Decay Characteristics of Primary Fluorescence from Live Bacteria

1987 ◽  
Vol 41 (2) ◽  
pp. 234-241 ◽  
Author(s):  
R. A. Dalterio ◽  
W. H. Nelson ◽  
D. Brut ◽  
J. F. Sperry ◽  
J. F. Tanguay ◽  
...  
Keyword(s):  
Steady State ◽  
Pseudomonas Fluorescens ◽  
Emission Spectra ◽  
Fluorescence Decay ◽  
Quantum Yields ◽  
Complex Nature ◽  
Fluorescence Lifetimes ◽  
Excitation Spectra ◽  
Primary Fluorescence ◽  
Rapid Characterization

The intrinsic steady-state fluorescence and fluorescence decay of Staphylococcus epidermidis, Pseudomonas fluorescens, Enterobacter cloacae, Escherichia coli, and Bacillus subtilis have been observed. Excitation spectra were obtained while emission at 430, 455, 487 and 514 nm was being monitored. Emission spectra were obtained with the use of excitation wavelengths of 340, 365, 405, 430 and 460 nm. Fluorescence lifetimes were measured at 430, 487, and 514 nm while selective excitation was caused at 340, 405, and 430 nm. The complex nature of the excitation and emission spectra reflects the presence of a number of different fluorophores. Attempts have been made to describe portions of the bacterial fluorescence in terms of the measured fluorescence properties including lifetimes of molecular components known for their widespread occurrence in bacteria and their relatively high quantum yields. Candidate fluorophores which have been considered include the pteridines, the structurally related flavins, and the pyridine coenzymes. The observation that characteristic sets of lifetimes have been obtained for each organism suggests that measurements of fluorescence lifetimes may be helpful in the rapid characterization of bacteria. Results are especially definitive in cases such as Pseudomonas fluorescens, where one marker fluorophore, a pteridine, is produced in large amounts.

scholarly journals Aniline Derived Bis-Schiff Base - Analytical Reagent for the Assay of Fe (III)

2018 ◽  
Vol 69 (11) ◽  
pp. 3097-3099
Author(s):  
Gladiola Tantaru ◽  
Nela Bibire ◽  
Alina Diana Panainte ◽  
Madalina Vieriu ◽  
Mihai Apostu
Keyword(s):  
Schiff Base ◽  
Quantitative Determination ◽  
Absorption Maximum ◽  
Pharmaceutical Products ◽  
Stable Complex ◽  
Complexation Reaction ◽  
Ich Guidelines ◽  
Analytical Reagent ◽  
Detection And Quantification

A new spectrophotometric method for the quantitative determination of Fe (III) was established based on the complexation reaction with a new bis-Schiff base, 4,4`-methylenebis-salicylidene aniline, when a stable complex with an absorption maximum at 520 nm was obtained. The conditions of the complexation reaction were established and the method was validated according to ICH guidelines in terms of linearity, accuracy, precision of the method and the limits of detection and quantification were determined. The method was applied with good results for the quantitative determination of Fe (III) in pharmaceutical products.

scholarly journals Utilizing Selective Chlorination to Synthesize New Triangulenium Dyes with Redshifted Absorption and Emission

2020 ◽  
Author(s):  
Jesper Dahl Jensen ◽  
Niels Bisballe ◽  
Laura Kacenauskaite ◽  
Maria Storm Thomsen ◽  
Bo W. Laursen
Keyword(s):  
Core Structure ◽  
Quantum Yields ◽  
Electrophilic Aromatic Substitution ◽  
Fluorescence Lifetimes ◽  
Aromatic Substitution ◽  
Compound A ◽  
Absorption And Emission ◽  
High Quantum ◽  
Selective Chlorination

Access to functionalization of a new site in the triangulenium core structure has been achieved at an early stageby chlorination with N-chlorosuccinimide (NCS), giving rise to two new triangulenium dyes (<b>1</b> and <b>2</b>). By introducing the chlorine functionalities in the acridinium precursor, positions complementary to those previously accessed by electrophilic aromatic substitution of the dyes are functionalized. The chlorination is highly selective giving only one regioisomer for both mono- and dichlorination. For the monochlorinated acridinium compound a highly selective ring-closing reaction was discovered generating only a single regioisomer of the cationic [4]helicene product. This discovery aspired further investigations into the mechanism of [4]helicene formation and to the first isolation of the previously proposed intermediate of the two-step S<sub>N</sub>Ar reaction key to all aza bridged triangulenium and helicenium synthesis. The fully ring closed mono- and di-chlorinated triangulenium dyes <b>1 </b>and <b>2</b> show a redshift in absorption and emission relative to the non-chlorinated analogues, while still maintaining relative high quantum yields of 36% and 41 %, and long fluorescence lifetimes of 15 ns and 16 ns, respectively.

scholarly journals Styryl-based new organic chromophores bearing free amino and azomethine groups: synthesis, photophysical, NLO, and thermal properties

2020 ◽  
Vol 16 ◽  
pp. 2282-2296
Author(s):  
Anka Utama Putra ◽  
Deniz Çakmaz ◽  
Nurgül Seferoğlu ◽  
Alberto Barsella ◽  
Zeynel Seferoğlu
Keyword(s):  
Schiff Base ◽  
Schiff Bases ◽  
Free Amino ◽  
Second Harmonic ◽  
Photophysical Properties ◽  
Bathochromic Shift ◽  
Visible Region ◽  
Amino Derivative ◽  
Quantum Yields ◽  
Thermal Stabilities

Herein we report the synthesis and characterization of a new series of styryl-based push-pull dyes containing a free amino group and their Schiff base derivatives. The dyes include the dicyanomethylene group as an acceptor and different para-substituted alkylamines as donors. Morever as a proton-sensitive group a pyridin-2-yl substituent was attached to the para-position of the phenyl moiety in both series of compounds. The photophysical properties of the dyes were examined in various solvents with different polarities and showed absorption in the visible region and green-red emission with low quantum yields. The absorption and the emission maxima were shifted bathocromically by increasing the solvent’s polarity. However, there was no correlation with the polarity parameters of the solvents. The pH-sensitive properties of all prepared Schiff bases were examined against TBAOH in DMSO, via deprotonation of the OH group in the salicylidene moiety and their reverse protonation was also investigated using TFA. The Schiff bases exhibited a bathochromic shift upon the addition of TBAOH to their solutions in DMSO. Therefore, they showed potential to be utilized as colorimetric and luminescence pH sensors. The second-order nonlinear optical (NLO) responses of the dyes were measured by the electric field-induced second harmonic (EFISH) generation method. The highest μβ values were obtained for the dyes bearing the julolidine donor as 1430 × 10−48 esu (for free amino derivative) and 1950 × 10−48 esu (for Schiff base derivative), respectively. The structural and electronic properties of the dyes as well as their NLO properties were further studied using DFT calculations. The thermal stabilities of all dyes were evaluated by thermogravimetric analysis (TGA). The TGA data showed that all dyes were thermally stable up to 250 °C.

Structural Factors Determining the Absorption Spectrum of Channelrhodopsins: A Case Study of the Chimera C1C2

2020 ◽  
Author(s):  
Suliman Adam ◽  
Christian Wiebeler ◽  
Igor Schapiro
Keyword(s):  
Hydrogen Bonding ◽  
Absorption Spectrum ◽  
Schiff Base ◽  
Absorption Maximum ◽  
Rational Design ◽  
Blue Shift ◽  
Spectral Shift ◽  
Structural Basis ◽  
Neural Cells ◽  
Structural Factors

Channelrhodopsins are photosensitive proteins that trigger flagella motion in single cell algae and have been successfully utilized in optogenetic applications. In optogenetics light is used to activate neural cells in living organisms, which can be achieved by exploiting the ion channel signaling of channelrhodopsins. Tailoring channelrhodopsins for such applications includes the tuning of the absorption maximum. In order to establish rational design and to obtain a desired spectral shift, a basic understanding of the absorption spectrum is required. We have studied the chimera C1C2 as a representative of this protein family and the first member with an available crystal structure. For this purpose, we sampled the conformations of C1C2 using QM/MM molecular dynamics, and subjected the resulting snapshots of the trajectory to excitation energy calculations using ADC(2) and simplified TD-DFT. In contrast to previous reports, we found that different hydrogen-bonding networks—involving the retinal protonated Schiff base, the putative counterions E162 and D292 as well as water molecules—had only a small impact on the absorption spectrum. However, in case of deprotonated E162 increasing the distance to the Schiff base hydrogen-bonding partner led to a systematic blue shift. The β-ionone ring rotation was identified as another important contributor. Yet the most important factor was found to be the bond length alternation and bond order alternation that were linearly correlated to the absorption maximum by up to 62 % and 82 %, respectively. We ascribe this novel insight into the structural basis of the absorption spectrum to our enhanced protein setup that includes membrane embedding as well as long and extensive sampling.

Synthesis and photophysical properties of fluorophore-labeled abscisic acid

2000 ◽  
Vol 78 (7) ◽  
pp. 963-974 ◽  
Author(s):  
X Hou ◽  
S R Abrams ◽  
J J Balsevich ◽  
N Irvine ◽  
T Norstrom ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Fluorescence Probe ◽  
Photophysical Properties ◽  
Fluorescence Decay ◽  
Quantum Yields ◽  
Excitation Spectra ◽  
Fluorescence Excitation ◽  
Fluorescence Quantum Yields ◽  
Fluorescence Excitation Spectra

The 8'-benzophenone, 8'-dansylhydrazone, 3'-S-(2-ethyldansylamide), and 3'-S-acetamidofluorescein derivatives of the plant hormone abscisic acid (ABA) have been synthesized for use in photoaffinity labeling (the benzophenone derivative) or fluorescence probe experiments and have been spectroscopically characterized. One of the three fluorescent compounds, the 3'-tethered fluorescein derivative, exhibits spectroscopic and photophysical properties which indicate that it could be an excellent fluorescent probe of ABA interactions in vivo. The 3'-tethered fluorescein and ABA moieties do not interact strongly, so that the fluorescence properties of the fluorescein-labelled hormone are very similar to those of fluorescein itself. Measurements of the absorption, emission, and fluorescence excitation spectra, fluorescence quantum yields, and fluorescence decay parameters of this derivative as a function of pH indicate that the photophysics is dominated by ground and excited state prototropic equilibria involving only the fluorescein moiety. The fluorescein dianion is the only significant absorber and emitter at pH > 6.7, whereas only the cation absorbs and emits at pH < 0. In the intervening pH range, strong emission from the monoanion and weak emission from two neutral species, tentatively assigned to the zwitterion and the lactone of the fluorescein moiety, is observed.Key words: abscisic acid, fluorescein, synthesis, photophysics.

Investigation of Spectra and Ionization Constants of 8-Alkyl Pterins by Fluorimetry

Pteridines ◽  
1993 ◽  
Vol 4 (1) ◽  
pp. 32-38 ◽  
Author(s):  
Soon-Seog Jeong ◽  
Paul Wormell ◽  
Jill E. Gready
Keyword(s):  
Fluorescence Intensity ◽  
Dissociation Constants ◽  
Ionization Constants ◽  
Quantum Yields ◽  
Excitation Spectra ◽  
Pyrazine Ring ◽  
Solvent Dependence ◽  
Fluorescence Characteristics ◽  
Wavelength Absorption ◽  
20 Nm

SummarySpectrofluorimetric studies of the properties of 8-alkyl-pterins arc reported, together with preliminary results on the use of fluorimetry in studying the binding of these new mechanism-based substrates to dihydrofolate reductase (DHFR). The compounds fluoresce strongly in the 460-490 nm range with emission maxima for the cations being blue-shifted by ~5-20 nm. Quantum yields for the cations of ~0.14-0.23 have been measured. pKa values for the basic ionization of ~5.3-5.7 have been determined from fits to both the emission and excitation spectra using sample concentrations in the 1-2 µM range. Although the UV /vis spectra between pH 0 and 4 show little change. there were significant sigmoidal changes in fluorescence intensity which could he fitted to apparent ionization constant values of ~1.4-1.0 with fluorescence intensity decaying to almost zero at pH 0. Studies of the fluorescence for the cations and neutral forms in organic acids and polar organic solvents respectively indicate little solvent dependence. Spectral modelling using the CNDO/S-CI method confirms that the spectra are due to π→π* transitions. and that the long wavelength absorption hand of ~400 nm is due to transitions involving mostly the pyrazine ring and N1. Significant differences in emission wavelength and intensity between 6-methyl- and 7 -methyl-8-alkyl-pterins have been characterised with the latter exhibiting fluorescence stronger by a factor of ~2 and blue-shifted by ~20 nm which arc readily detectable visually: aqua-blue and green fluorescence respectively for the 7-methyl and 6-methyl isomers. From this understanding of the fluorescence properties of 8-alkyl-pterins and their dependence on charge state and pyrazine-ring substituents, and taking into account the fluorescence characteristics of DHFR and cofactor NADP. we have assessed the potential of fluorimetric experiments for studying both pterin dissociation constants and the form of enzyme-bound pterin

scholarly journals Influence of the melting temperature on the measurement of the mass concentration and size distribution of black carbon in snow

2016 ◽  
Vol 9 (4) ◽  
pp. 1939-1945 ◽  
Author(s):  
Takeshi Kinase ◽  
Kazuyuki Kita ◽  
Yoshimi Tsukagawa-Ogawa ◽  
Kumiko Goto-Azuma ◽  
Hiroto Kawashima
Keyword(s):  
Size Distribution ◽  
Black Carbon ◽  
Melting Temperature ◽  
Mass Concentration ◽  
Melting Time ◽  
Snow Sample ◽  
Melted Snow ◽  
Different Temperatures ◽  
Long Time ◽  
Snow Samples

Abstract. The influence of temperature and time of snow sample melting on the measurement of mass concentration and size distribution of black carbon (BC) in snow was evaluated experimentally. In the experiments, fresh (Shirouma) and aged (Hakusan) snow samples were melted at different temperatures or at different time lengths, and the BC mass concentration and size distribution in the melted snow samples were measured using a nebulizer and a single-particle soot photometer (SP2). In the experiment where melting temperature was varied, the BC mass concentration in the liquid decreased at a melting temperature of 70 °C. This decrease was 8.0 % for the Shirouma sample and 46.4 % for the Hakusan sample and depended on BC particle size, with a significant decrease found at BC diameters less than 350 nm. A similar decrease in BC mass concentration was found when the Hakusan snow sample that had been melted at 5 °C was heated to 70 °C. The experiment in which melting time was varied indicated that BC mass concentration in the liquid did not change for the Shirouma sample but decreased significantly with a longer melting time for the Hakusan sample (38.6 %). These results indicate that melting of snow samples at high temperatures or over long time periods can significantly affect the measurement of BC mass and its size distribution, especially for aged snow samples.

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