scholarly journals Solid state fluorescence of proteins in high throughput mode and its applications

F1000Research ◽  
2019 ◽  
Vol 2 ◽  
pp. 82
Author(s):  
Saurabh Gautam ◽  
Munishwar N Gupta
Keyword(s):  
Solid State ◽  
High Throughput ◽  
Fluorescence Spectra ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Simple Method

Direct comparison between fluorescence spectra of a sample in solution and solid state form is valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples in a high-throughput format. The 96-well plate used in our studies, was coated black from all the sides and the excitation and emission paths are identical and are from the top of the well. These two features minimize scatter and provide fairly noise free spectra. Even then the fluorescence intensity may be dependent upon many factors such as the extent of protein aggregation, morphology and sizes of the protein particles. Hence, (changes in) λmax emission may be a more reliable metric in the case of fluorescence spectra of proteins in the solid state. However, any large changes in the intensity could indicate changes in the microenvironment of the fluorophore. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form, and the lyophilized powder form. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. We also analyzed fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP. These findings pointed towards the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.

scholarly journals Solid state fluorescence of proteins in high throughput mode and its applications

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 82
Author(s):  
Saurabh Gautam ◽  
Munishwar N Gupta
Keyword(s):  
Solid State ◽  
High Throughput ◽  
Fluorescence Spectra ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Red Shift ◽  
Solid Samples ◽  
Fluorescence Emission Spectra ◽  
Simple Method

A simple method to determine fluorescence emission spectra of proteins in solid state is described. The available commercial accessories can only accommodate solid samples and hence do not allow a direct comparison between fluorescence spectra of a sample in solution and solid state form. Such comparisons are valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). The commercially available accessories also do not allow working in a high throughput mode. We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples. This method works with a 96-well plate format. It enables the comparison of fluorescence spectra of a sample in a solid state with solution spectra, using comparable quantities of protein. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form and the lyophilized powder form. α-Chymotrypsin in solution showed a λmax of 335 nm while a high-activity preparation of the same enzyme for non-aqueous media, known as enzyme precipitated and rinsed with propanol (EPRP), showed an increase in the intensity of the fluorescence emission spectra. However, there was a small red shift of 2 nm (λmax of 337 nm) in contrast to lyophilized powder which showed a λmax of 328 nm. This is due to a difference in the tertiary structure of the protein as well as the microenvironment of aromatic residues between the two preparations. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. The relative fluorescence intensity of lyophilized GFP powder was decreased significantly to 17% as compared to GFP in solution, and showed a red shift of 4 nm in the emission λmax. It was found that fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP was significantly diminished. This indicated the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.

scholarly journals Efficiency of Energy Transfer for TiO2 Nanoparticles with Fluorescein Dye

2019 ◽  
Vol 29 (4) ◽  
pp. 134
Author(s):  
Mahasin F. Hadi Al-Kadhemy ◽  
Asrar Abdulmunem ◽  
Husam Sabeeh Al-Arab
Keyword(s):  
Energy Transfer ◽  
Tio2 Nanoparticles ◽  
Absorption Spectra ◽  
Water Solution ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Emission Spectra ◽  
Major Effect ◽  
Fluorescence Emission Spectra

For different amount of masses of TiO2 nanoparticles in dye solution, absorption and Fluorescence profiles of the suspension for TiO2 nanoparticles with Fluorescein (F) in distilled water solution, has been explored. An absorption spectra enhancement were detected for changed amount of masses, which specifies that the doping with TiO2 nanoparticles has a major effect on the dye absorption spectra. Contrarily, all fluorescence emission spectra for the dye, were quenched as TiO2 nanoparticles amount of masses increases because of Förster resonance energy transfer (FRET).

scholarly journals Luminescent Hybrid Materials Based on Curcumin Derivatives Embedded in Palygorskite

2018 ◽  
Vol 55 (1) ◽  
pp. 63-67
Author(s):  
Monica Florentina Raduly ◽  
Valentin Raditoiu ◽  
Alina Raditoiu ◽  
Luminita Eugenia Wagner ◽  
Viorica Amariutei ◽  
...  
Keyword(s):  
Hybrid Materials ◽  
Fluorescence Spectra ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Hydroxyl Groups ◽  
Fluorescent Properties ◽  
Fluorescence Emission Spectra ◽  
Curcumin Derivatives ◽  
Absorption Studies ◽  
Inorganic Matrix

The seven curcumin derivatives were deposited on palygorskite in order to obtain hybrid materials. The fluorescence emission spectra of the obtained materials show a decrease in fluorescence intensity relative to the respective dyes, due to the environments around the dyestuff molecules created in the host matrices. Absorption studies show the best adsorption on the inorganic matrix, for the compounds with the hydroxyl groups. Correlating fluorescence spectra of hybrid materials with the results for absorption spectra of the dyes adsorbtion on the surface of the clay lead to the conclusion that a high percentage of the adsorbed dye had the effect of fluorescence quenching. Thus, it was confirmed that the fluorescent properties of hybrid materials depend on the interactions established between the fluorescent dyestuff and the inorganic network.

Note on Solid State Fluorescence Emission of Ochratoxins A and B on Silica Gel

1970 ◽  
Vol 53 (4) ◽  
pp. 696-697
Author(s):  
Fun Sun Chu
Keyword(s):  
Solid State ◽  
Ochratoxin A ◽  
Silica Gel ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Ochratoxin B ◽  
Solid State Fluorescence

Abstract The fluorescence emission spectra of ochratoxins A and B on silica gel have been determined. The emission maxima for both toxins were found to be around 475 nm, with excitation maxima at 340 nm for ochratoxin A and 325 nm for ochratoxin B.

scholarly journals Discovery of Enzyme Modulators via High-Throughput Time-Resolved FRET in Living Cells

2014 ◽  
Vol 19 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Simon J. Gruber ◽  
Razvan L. Cornea ◽  
Ji Li ◽  
Kurt C. Peterson ◽  
Tory M. Schaaf ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Chemical Library ◽  
Time Resolved ◽  
Endoplasmic Reticulum Calcium ◽  
Green Fluorescent

We have used a “two-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Alteration of fluorescent protein spectroscopic properties upon cryoprotection

2012 ◽  
Vol 68 (11) ◽  
pp. 1578-1583 ◽  
Author(s):  
David von Stetten ◽  
Gaëlle O. Batot ◽  
Marjolaine Noirclerc-Savoye ◽  
Antoine Royant
Keyword(s):  
Ethylene Glycol ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Spectroscopic Properties ◽  
Protein Crystal ◽  
Structural Effects ◽  
Noticeable Effect ◽  
Fluorescence Emission Spectra

Cryoprotection of a protein crystal by addition of small-molecule compounds may sometimes affect the structure of its active site. The spectroscopic and structural effects of the two cryoprotectants glycerol and ethylene glycol on the cyan fluorescent protein Cerulean were investigated. While glycerol had almost no noticeable effect, ethylene glycol was shown to induce a systematic red shift of the UV–vis absorption and fluorescence emission spectra. Additionally, ethylene glycol molecules were shown to enter the core of the protein, with one of them binding in close vicinity to the chromophore, which provides a sound explanation for the observed spectroscopic changes. These results highlight the need to systematically record spectroscopic data on crystals of light-absorbing proteins and reinforce the notion that fluorescent proteins must not been seen as rigid structures.

A Novel Method for the Preparation of Nanoaggregates of Methoxy Polyethyleneglycol Linked Chitosan

2006 ◽  
Vol 6 (9) ◽  
pp. 2867-2873 ◽  
Author(s):  
Anandrao R. Kulkarni ◽  
Yu-Hsin Lin ◽  
Hsiang-Fa Liang ◽  
Wei-Chun Chang ◽  
Wesley Wei-Wen Hsiao ◽  
...  
Keyword(s):  
Fluorescence Emission ◽  
Emission Spectra ◽  
Gel Permeation Chromatography ◽  
Aqueous Solubility ◽  
Degree Of Substitution ◽  
Critical Aggregation Concentration ◽  
Fluorescence Emission Spectra ◽  
Simple Method ◽  
Ft Ir ◽  
Sensitive Property

In the study, methoxy polyethyleneglycol (MPEG) linked chitosan (PLC) with a different degrees of substitution were prepared using a novel yet simple method in the presence of formaldehyde in a solvent of formic acid and dimethylsulfoxide (DMSO). The obtained PLC was verified by the Fourier transformed infrared (FT-IR) and carbon nuclear magnetic resonance (13C-NMR) spectroscopy and by the gel permeation chromatography (GPC). The aqueous solubility of chitosan increased after chemically linking with MPEG and was found to depend on its degree of substitution. With a proper degree of substitution of MPEG on chitosan, PLC may undergo inter- and/or intra-molecular entanglements to produce nanoaggregates. The critical aggregation concentration (CAC) of PLC was determined by the fluorescence emission spectra of pyrene and was found to be 0.003 mg/ml. Measurements of the size distribution and zeta potential of the prepared nanoaggregates were carried out using a Zetasizer. The results suggested that as the degree of MPEG substitution increased, the size and polydispersity index of the prepared nanoaggregates decreased. The prepared nanoaggregates showed a pH-sensitive property and thus may be suitable for the development of drug delivery devices for tumors.

scholarly journals Study of the behavior of short chains in print paper subjected to a natural aging

2018 ◽  
Vol 31 (4) ◽  
pp. 58-62
Author(s):  
José Martínez Mendoza ◽  
Alejandra Nieto-Villena ◽  
José Angel De la Cruz-Mendoza ◽  
Gerardo Ortega-Zarzosa ◽  
Azdrubal Guerrero-Serrano
Keyword(s):  
Structural Stability ◽  
Ethyl Alcohol ◽  
Fluorescence Spectra ◽  
Fluorescence Emission ◽  
Natural Aging ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Paper Making ◽  
Intensity Changes

Degradation of paper through time can be measured by monitoring the intensity changes in the band located at 620 nm of its emission fluorescence spectra. The behavior of this band is closely related to the structural stability of the short cellulose chains. A fast degradation of these cellulose chains, mainly lignin, translates in the rapid denaturation of paper occurring at its first aging state. This work proposes a method to reincorporate short cellulose chains in the structure of aged paper through its immersion during 24 h in a solution based on ethyl alcohol and extracts of wood pieces. Results of fluorescence spectra measurements indicate that the short cellulose chains in the solution get embedded in the old paper, making the shape and intensity of the fluorescence emission spectra peaks of new and old paper almost indistinguishable. This behavior provides a desirable approach to analyze, preserve and restore aged paper documents.

scholarly journals Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

2016 ◽  
Vol 22 (3) ◽  
pp. 250-261 ◽  
Author(s):  
Tory M. Schaaf ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
David D. Thomas ◽  
Gregory D. Gillispie
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Unmixing ◽  
Fluorescence Emission Spectrum ◽  
Plate Reader

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

Synthesis and Optical Properties of some Novel Bis-1,3,4-Oxadiazoles

2015 ◽  
Vol 1104 ◽  
pp. 131-135
Author(s):  
Hua Liu ◽  
Lin Lu ◽  
Dun Jia Wang
Keyword(s):  
Optical Properties ◽  
Fluorescence Spectra ◽  
Fluorescence Emission ◽  
Chloroform Solution ◽  
Emission Spectra ◽  
Pyridine Derivatives ◽  
Fluorescence Emission Spectra ◽  
H Nmr ◽  
Uv Illumination ◽  
Oxadiazole Derivatives

Three 2,6-bis (1,3,4-oxadiazol-2-yl) pyridine derivatives were synthesized and their structures were conformed by1H NMR, FTIR, MS techniques and elemental analysis. The UV–Vis absorption and fluorescence emission spectra of these compounds were investigated in chloroform solution. The results showed that these bis-1,3,4-oxadiazole derivatives had broad and strong absorption at 278−302 nm in the UV–Vis spectra and exhibited fluorescence emission at 338 −367 nm under UV illumination in fluorescence spectra. It was found that the nature of the substituents at benzene ring in bis-1,3,4-oxadiazole derivatives had a significant impact on their photoluminescence behaviors.

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