Alteration of fluorescent protein spectroscopic properties upon cryoprotection

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.

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The dependence of electrophoretic and spectroscopic properties of the pyruvate dehydrogenase complex on mono- and divalent ions.

Acta Biochimica Polonica ◽

10.18388/abp.1994_4775 ◽

1994 ◽

Vol 41 (1) ◽

pp. 63-72

Author(s):

T Pawełczyk ◽

R A Easom ◽

M S Olson

Keyword(s):

Ionic Strength ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Fluorescence Emission ◽

Emission Spectra ◽

Spectroscopic Properties ◽

Uv Absorption ◽

Divalent Ions ◽

Fluorescence Emission Spectra ◽

Dehydrogenase Complex

The effects of various mono- and divalent ions on the pyruvate dehydrogenase complex (PDC) were investigated. To determine the radius of PDC under various conditions a two-dimensional agarose gel electrophoresis technique was used. The radius of PDC cross-linked with glutaraldehyde at ionic strength 0.04 M was calculated to be 22.0 +/- 0.1 nm. The presence of K+, Na+ or HPO4(2-) prevented changes in electromobility and of the calculated radius of PDC induced by alteration in ionic strength. The fluorescence emission spectra of PDC depended on the ionic strength and monovalent cations. The fluorescence intensity of PDC increased in the presence of 80 mM K+, and decreased in the presence of 80 mM Na+ with no shift in the emission maximum wavelength. Changes in the ionic strength to which PDC was exposed resulted in alteration of the UV absorption spectra in the 230 nm region. These alterations were prevented by HPO4(2-), whereas Na+ or K+ ions had no effect on the UV absorption spectrum of PDC.

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Comparative Analysis of Bacteriophytochrome Agp2 and Its Engineered Photoactivatable NIR Fluorescent Proteins PAiRFP1 and PAiRFP2

Biomolecules ◽

10.3390/biom10091286 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1286 ◽

Cited By ~ 1

Author(s):

Faez Iqbal Khan ◽

Fakhrul Hassan ◽

Razique Anwer ◽

Feng Juan ◽

Dakun Lai

Keyword(s):

Comparative Analysis ◽

Structural Changes ◽

Near Infrared ◽

Fluorescent Proteins ◽

Irradiation Time ◽

Fluorescence Emission ◽

Emission Spectra ◽

Md Simulations ◽

Fluorescence Emission Spectra ◽

Molecular Stability

Two photoactivatable near infrared fluorescent proteins (NIR FPs) named “PAiRFP1” and “PAiRFP2” are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAiRFP2 were more deviated than Agp2 from its native conformation and the hydrophobic regions that were buried in PAiRFP1 and PAiRFP2 core exposed to solvent molecules. The eigenvalues and the trace of covariance matrix were found to be high for PAiRFP1 (597.90 nm2) and PAiRFP2 (726.74 nm2) when compared with Agp2 (535.79 nm2). It was also found that PAiRFP1 has more sharp Gibbs free energy global minima than Agp2 and PAiRFP2. This comparative analysis will help to gain deeper understanding on the structural changes during the evolution of photoactivatable NIR FPs. Further work can be carried out by combining PCR-based directed mutagenesis and spectroscopic methods to provide strategies for the rational designing of these PAiRFPs.

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

F1000Research ◽

10.12688/f1000research.2-82.v2 ◽

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.

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Spectroscopic and Binding Properties of Near-Infrared Tricarbocyanine Dyes to Double-Stranded DNA

Applied Spectroscopy ◽

10.1366/0003702963906429 ◽

1996 ◽

Vol 50 (2) ◽

pp. 211-221 ◽

Cited By ~ 20

Author(s):

Yolanda Y. Davidson ◽

Bonnie M. Gunn ◽

Steven A. Soper

Keyword(s):

Near Infrared ◽

Fluorescence Emission ◽

Emission Spectra ◽

Spectroscopic Properties ◽

Fluorescence Emission Spectra ◽

State Aggregation ◽

Double Stranded Dna ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Spectrophotometric Titrations

The noncovalent binding and spectroscopic properties of several near-infrared tricarbocyanine dyes with respect to sonicated calf-thymus DNA are reported. The dyes investigated were diethylthiatricarbocyanine iodide (DTTCI), diethyloxatricarbocyanine iodide (DOTCI), and 1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide (HITCI), which are cationic and possess absorption maxima at 772, 695, and 750 nm, respectively, in DMSO. In buffered aqueous solutions, these dyes demonstrated extensive ground-state aggregation in aqueous solvents when compared to DMSO. In the presence of double-stranded DNA (dsDNA), the fluorescence emission spectra revealed enhancement ratios of bound-to-free dye ranging from 4.5 for DOTCI to 128 for DTTCI. Spectrophotometric titrations and Scatchard analyses of the dye-dsDNA complexes yielded nonlinear plots, suggestive of possible multiple binding sites on the DNA. Viscometric titrations of the complexes showed increased solution viscosities for DTTCI, consistent with an unraveling and lengthening of the dsDNA upon complexation. Fluorescence lifetime data of the dye-dsDNA complexes showed longer lifetimes exhibited by these dyes in the presence of the dsDNA compared with those in solutions with no DNA.

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Spectroscopic Properties of Polycyclic Aromatic Compounds. Part III: Fluorescence Emission and Quenching Behavior of Periodic Table Group 16 Hetero-Atom Derivatives

Applied Spectroscopy ◽

10.1366/0003702934066622 ◽

1993 ◽

Vol 47 (3) ◽

pp. 317-320 ◽

Cited By ~ 3

Author(s):

Sheryl A. Tucker ◽

William E. Acree ◽

Maximilian Zander ◽

Pierre Demerseman ◽

Jean-Pierre Buisson

Keyword(s):

Dynamic Range ◽

Fluorescence Emission ◽

Emission Spectra ◽

Spectroscopic Properties ◽

Polycyclic Aromatic Compounds ◽

Fluorescence Emission Spectra ◽

Polycyclic Aromatic Compound ◽

Polycyclic Aromatic ◽

Organic Nonelectrolyte ◽

Intensity Ratios

Fluorescence emission spectra are reported for benzo[b]naphtho[2,3d]furan, dinaphtho[l,2b:l′,2′d]furan, dinaphtho[2,lb:l′,2′d]furan, dibenzo[2,3:10,11]perylo[l,12bcd]furan, dibenzo[2,3:10,ll]perylo-(l,12bcd]thiophene, naphtho[l,8bc:5,4b′c′]dipyran (also called 1,6-dioxapyrene), and naphtha[l,8bc:4,5b′c′]dipyran (also called 1,8-dioxapyrene) in organic nonelectrolyte solvents of varying polarity. Results of these measurements indicate that dinaphtho[l,2b:l′,2′d]furan exhibits slight signs of probe character as evidenced by changing emission intensity ratios; however, the dynamic range was much too small to classify this molecule as a polycyclic aromatic compound probe. The effect of nitromethane and 1,2,4-trimethoxybenzene as selective quenching agents was also examined. Nitromethane was found to quench fluorescence emission of all the aforementioned compounds except benzo[b]naphtho[2,3d]furan.

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Spectroscopic and Structural Analysis of Cu2+-Induced Fluorescence Quenching of ZsYellow

Biosensors ◽

10.3390/bios10030029 ◽

2020 ◽

Vol 10 (3) ◽

pp. 29 ◽

Cited By ~ 1

Author(s):

In Jung Kim ◽

Yongbin Xu ◽

Ki Hyun Nam

Keyword(s):

Structural Analysis ◽

Fluorescence Quenching ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Fluorescence Emission ◽

Spectroscopic Properties ◽

Dependent Manner ◽

Quenching Analysis ◽

High Degree

Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu2+. During Cu2+-induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors.

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Optical Properties and Zinc Nitride Thin Films Prepared Using Magnetron Reactive Sputtering

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.940.11 ◽

2014 ◽

Vol 940 ◽

pp. 11-15

Author(s):

Jun Qin Feng ◽

Jun Fang Chen

Keyword(s):

Thin Films ◽

Band Gap ◽

Near Infrared ◽

Fluorescence Emission ◽

Emission Spectra ◽

Sem Images ◽

Fluorescence Emission Spectra ◽

Glass Substrates ◽

Zinc Nitride ◽

Direct Band

Zinc nitride films were deposited by ion sources-assisted magnetron sputtering with the use of Zn target (99.99% purity) on 7059 glass substrates. The films were characterized by XRD, SEM and EDS, the results of which show that the polycrystalline zinc nitride thin film can be grown on the glass substrates, the EDS spectrum confirmed the chemical composition of the films and the SEM images revealed that the zinc nitride thin films have a dense structure. Ultraviolet-visible-near infrared spectrophotometer was used to study the transmittance behaviors of zinc nitride thin films, which calculated the optical band gap by Davis Mott model. The results of the fluorescence emission spectra show the zinc nitride would be a direct band gap semiconductor material.

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Stress and Phase Transformation Phenomena in Oxide Films: Real-Time Spectroscopic Measurements

MRS Proceedings ◽

10.1557/proc-271-319 ◽

1992 ◽

Vol 271 ◽

Cited By ~ 3

Author(s):

Gregory J. Exarhos ◽

Nancy J. Hess

Keyword(s):

Phase Transformation ◽

Applied Pressure ◽

Oxide Films ◽

Fluorescence Emission ◽

Emission Spectra ◽

Film Deposition ◽

Optical Methods ◽

Fluorescence Emission Spectra ◽

Line Intensities ◽

Deposition Parameters

ABSTRACTIn situ optical methods are reviewed for characterization of phase transformation processes and evaluation of residual stress in solution-deposited metastable oxide films. Such low density films most often are deposited as disordered phases making them prone to crystallization and attendant densification when subjected to increased temperature and/or applied pressure. Inherent stress imparted during film deposition and its evolution during the transformation are evaluated from phonon frequency shifts seen in Raman spectra (TiO2) or from changes in the laser-induced fluorescence emission spectra for films containing rare earth (Sm+3:Y3Al5O12) or transition metal (Cr+3 :Al2O3) dopants. The data in combination with measured increases in line intensities intrinsic to the evolving phase are used to follow crystallization processes in thin films. In general, film deposition parameters are found to influence the crystallite ingrowth kinetics and the magnitude of stress and stress relaxation in the film during the transformation. The utility of these methods to probe crystallization phenomena in oxide films will be addressed.

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