scholarly journals Abstract P-17: Photophysical Properties of Freely Diffusing and Immobilized Fluorescent Conjugate Based on Calcium-Binding Protein Recoverin and Alexa647 Dye

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S18-S19
Author(s):  
Ilia Zykov ◽  
Ivan Maslov ◽  
Evgeni Zernii ◽  
Sergei Permyakov ◽  
Thomas Gensch ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Photophysical Properties ◽  
Fluorescence Emission ◽  
Specific Protein ◽  
Fluorescence Lifetime Imaging ◽  
Visual Transduction ◽  
The Matrix ◽  
Rhodopsin Kinase ◽  
Induced Changes

Background: Recoverin is a calcium sensor membrane-associated protein that inhibits rhodopsin kinase thereby participating in the regulation of visual transduction. Here we examined calcium-induced conformational changes in recoverin conjugated with fluorescent dye Alexa647. Methods: Photophysical properties of immobilized and freely diffusing recoverin were investigated using fluorescence lifetime imaging microscopy and fluorescence emission spectroscopy. In solution, the formation and dissociation of the Ca2+-recoverin complex manifested as changes in Alexa647 spectra and the lifetime. In contrast, immobilization of recoverin on the microscopy glass via biotin–NeutrAvidin–biotinylated polyethylene glycol (PEG) tether inhibited changes in fluorescent signal. That can be provided by PEG as it prevented the calcium-induced changes in spectrum and lifetime of recoverin-bound Alexa647 in solution. The use of another immobilization facilitator, bovine serum albumin (BSA), did not affect calcium-induced changes in fluorescence of the conjugate in solution but produced the matrix, which was ineffective in recoverin immobilization. Results: Microscale thermophoresis demonstrated that biotinylated recoverin interacted with NeutrAvidin in solution indicating that immobilization affinity depended mainly on the geometry of the glass coating surface. Conclusion: Our results highlight the challenge of specific protein immobilization that does not affect protein functionality. By the example of recoverin, we showed that the employment of two common immobilization facilitators, PEG and BSA, yielded surfaces with different space geometry, which differently affect NeutrAvidin-based immobilization affinity as well as Ca2+-dependent conformational changes of the biotinylated protein.

scholarly journals Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S15-S15
Author(s):  
Anatolii Belousov ◽  
Ivan Maslov ◽  
Polina Khorn ◽  
Alexander Mishin ◽  
Mikhail Baranov ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Dyes ◽  
Conformational Dynamics ◽  
Emission Spectra ◽  
Spectroscopic Observation ◽  
Visual Signals ◽  
Low Light ◽  
Ef Hand ◽  
Rhodopsin Kinase ◽  
Induced Changes

Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.

Pinocytotic-Like Forms of Mitochondria From Rat Anterior Pituitary

1968 ◽  
Vol 26 ◽  
pp. 146-147
Author(s):  
Burton B. Silver
Keyword(s):  
Electron Micrographs ◽  
Conformational Changes ◽  
Anterior Pituitary ◽  
Dynamic State ◽  
Surrounding Cell ◽  
The Matrix ◽  
The Moment ◽  
Cell Medium ◽  
Sectioned Tissue ◽  
State Of Activity

Sectioned tissue rarely indicates evidence of what is probably a highly dynamic state of activity in mitochondria which have been reported to undergo a variety of movements such as streaming, divisions and coalescence. Recently, mitochondria from the rat anterior pituitary have been fixed in a variety of configurations which suggest that conformational changes were occurring at the moment of fixation. Pinocytotic-like vacuoles which may be taking in or expelling materials from the surrounding cell medium, appear to be forming in some of the mitochondria. In some cases, pores extend into the matrix of the mitochondria. In other forms, the remains of what seems to be pinched off vacuoles are evident in the mitochondrial interior. Dense materials, resembling secretory droplets, appear at the junction of the pores and the cytoplasm. The droplets are similar to the secretory materials commonly identified in electron micrographs of the anterior pituitary.

Immunogold localization of calmodulin in root caps of the maize Cultivar Merit

1990 ◽  
Vol 48 (3) ◽  
pp. 674-675
Author(s):  
P.T. Nguyen ◽  
C. Uphoff ◽  
C.L. Stinemetz
Keyword(s):  
Calcium Binding ◽  
Calcium Transport ◽  
Primary Role ◽  
Immunogold Localization ◽  
Root Tips ◽  
Electrical Currents ◽  
Considerable Evidence ◽  
Maize Cultivar ◽  
Gravity Response ◽  
Induced Changes

Considerable evidence suggest that the calcium-binding protein calmodulin (CaM) may mediate calcium action and/or transport important in the gravity response of plants. Calmodulin is present in both shoots and roots and is capable of regulating calcium transport in plant vesicles. In roots calmodulin is concentrated in the tip, the gravisensing region of the root; and is reported to be closely associated with amyloplasts, organelles suggested to play a primary role in gravi-perception. Inhibitors of CaM such as chlorpromazine, calmidazolium, and compound 48/80 interfere with the gravitropic response of both snoots and roots. The magnitude of the inhibition corresponded well with the extent to which the drug binds to endogenous CaM. Compound 48/80 and calmidazolium block gravi-induced changes in electrical currents across root tips, a phenomenon thought to be associated with the sensing of the gravity stimulus.In this study, we have investigated the subcellular distribution of CaM in graviresponsive and non-graviresponsive root caps of the maize cultivar Merit.

scholarly journals Exciton Coupling and Conformational Changes Impacting the Excited State Properties of Metal Organic Frameworks

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4230
Author(s):  
Andreas Windischbacher ◽  
Luca Steiner ◽  
Ritesh Haldar ◽  
Christof Wöll ◽  
Egbert Zojer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Photophysical Properties ◽  
Metal Organic Framework ◽  
Metal Organic Frameworks ◽  
Red Shift ◽  
Light Emitting ◽  
Light Emitting Devices ◽  
Exciton Coupling ◽  
Metal Organic ◽  
Crystalline Metal

In recent years, the photophysical properties of crystalline metal-organic frameworks (MOFs) have become increasingly relevant for their potential application in light-emitting devices, photovoltaics, nonlinear optics and sensing. The availability of high-quality experimental data for such systems makes them ideally suited for a validation of quantum mechanical simulations, aiming at an in-depth atomistic understanding of photophysical phenomena. Here we present a computational DFT study of the absorption and emission characteristics of a Zn-based surface-anchored metal-organic framework (Zn-SURMOF-2) containing anthracenedibenzoic acid (ADB) as linker. Combining band-structure and cluster-based simulations on ADB chromophores in various conformations and aggregation states, we are able to provide a detailed explanation of the experimentally observed photophysical properties of Zn-ADB SURMOF-2: The unexpected (weak) red-shift of the absorption maxima upon incorporating ADB chromophores into SURMOF-2 can be explained by a combination of excitonic coupling effects with conformational changes of the chromophores already in their ground state. As far as the unusually large red-shift of the emission of Zn-ADB SURMOF-2 is concerned, based on our simulations, we attribute it to a modification of the exciton coupling compared to conventional H-aggregates, which results from a relative slip of the centers of neighboring chromophores upon incorporation in Zn-ADB SURMOF-2.

scholarly journals Photophysical properties and fluorosolvatochromism of D–π–A thiophene based derivatives

RSC Advances ◽  
2020 ◽  
Vol 10 (71) ◽  
pp. 43459-43471
Author(s):  
Hussain A. Z. Sabek ◽  
Ahmed M. M. Alazaly ◽  
Dina Salah ◽  
Hesham S. Abdel-Samad ◽  
Mohamed A. Ismail ◽  
...  
Keyword(s):  
Photophysical Properties ◽  
Fluorescence Emission ◽  
Time Resolved ◽  
Time Resolved Spectroscopy ◽  
Solvation Models

Solvation-dependent photophysical properties of two push–pull thiophene-based compounds with donor–π–acceptor (D–π–A) structures were investigated using absorption, fluorescence emission and time resolved spectroscopy, and supported by different solvation models.

scholarly journals Photophysical Properties of Protoporphyrin IX, Pyropheophorbide-a, and Photofrin® in Different Conditions

2021 ◽  
Vol 14 (2) ◽  
pp. 138
Author(s):  
Bauyrzhan Myrzakhmetov ◽  
Philippe Arnoux ◽  
Serge Mordon ◽  
Samir Acherar ◽  
Irina Tsoy ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Photophysical Properties ◽  
Protoporphyrin Ix ◽  
Fluorescence Emission ◽  
Solvent Polarity ◽  
Visible Absorption ◽  
Light Dosimetry ◽  
Time Resolved ◽  
Pyropheophorbide A ◽  
Influence Of Solvent

Photodynamic therapy (PDT) is an innovative treatment of malignant or diseased tissues. The effectiveness of PDT depends on light dosimetry, oxygen availability, and properties of the photosensitizer (PS). Depending on the medium, photophysical properties of the PS can change leading to increase or decrease in fluorescence emission and formation of reactive oxygen species (ROS) especially singlet oxygen (1O2). In this study, the influence of solvent polarity, viscosity, concentration, temperature, and pH medium on the photophysical properties of protoporphyrin IX, pyropheophorbide-a, and Photofrin® were investigated by UV-visible absorption, fluorescence emission, singlet oxygen emission, and time-resolved fluorescence spectroscopies.

scholarly journals Mutations in calphotin, the gene encoding a Drosophila photoreceptor cell-specific calcium-binding protein, reveal roles in cellular morphogenesis and survival.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 413-421 ◽  
Author(s):  
Y Yang ◽  
D Ballinger
Keyword(s):  
Calcium Binding ◽  
Photoreceptor Cell ◽  
Cell Structure ◽  
Specific Protein ◽  
Cell Organelle ◽  
Cell Type ◽  
Gene Encoding ◽  
Cellular Morphogenesis ◽  
Transgenic Flies ◽  
Drosophila Photoreceptor

Abstract Calphotin is a Drosophila photoreceptor cell-specific protein expressed very early in eye development, at the time when cell-type decisions are being made. Calphotin is a very hydrophobic and proline-rich protein which lacks obvious transmembrane domains. The cDNA encoding Calphotin was mapped to a region removed by a set of existing chromosomal deletions. Mutations that alter photoreceptor cell structure and development were isolated that fail to complement these deletions. These mutations fall into two classes. Class I mutations alter the structure of the rhabdomere, a photoreceptor cell organelle specialized for phototransduction. Class II mutations have rough eyes, due to misorientation of the rhabdomeres and photoreceptor cell death. Transformation rescue of these phenotypes in transgenic flies bearing calphotin genomic DNA indicates that both classes of mutations are in the calphotin gene. Analysis of these mutations suggest that Calphotin plays important roles in both rhabdomere development and in photoreceptor cell survival.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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