DFT study of the interaction between the conjugated fluorescein and dabcyl system, using fluorescene quenching method

Alvarado-González, Mónica; Gallo, Marco; López-Albarrán, Pablo; Flores-Holgúın, Norma; Glossman‐Mitnik, Daniel

doi:10.1007/s00894-012-1413-4

Cited by 10 publications

(10 citation statements)

References 18 publications

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“…Dabcyl is well-known as a quencher in FRET pairs with Cf [ 37 ]. This feature may represent a challenge since during flow cytometry the detected fluorescence can be emitted mainly from cleaved peptides only, while non-cleaved peptides are not detected due to quenching.…”

Section: Resultsmentioning

confidence: 99%

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers

et al. 2022

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Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure–activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

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Section: Resultsmentioning

confidence: 99%

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers

et al. 2022

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“…The mTFP-PNA:Beacon:PNA2 assembly (2.5 μM mTFP-PNA:27 μM DNA; beacon at saturating conditions) exhibited a 59 ± 4% energy transfer over the range of 490–510 nm relative to a control sample of mTFP:DNA beacon. Control samples consisting of mTFP:DNA beacon resulted in up to 40% intensity decrease when added to unligated mTFP, which can be ascribed to inner filter effects arising from the highly colored solution seen with the highest beacon concentrations and slight quenching effect of Dabcyl when the fluophores are in 7 nm distance in open form ( R 0 : 4.6 nm). Similar effects were not observed with dilute solutions (e.g., 2.7 μM DNA beacon).…”

Section: Resultsmentioning

confidence: 99%

PNA-Induced Assembly of Fluorescent Proteins Using DNA as a Framework

2013

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Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein-PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)-peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA's assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

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“…The linker was approximated as a semiflexible cylinder with width w link ¼ 0.45 nm and lengths L link ¼ 1.65, 1.35, and 2.05 nm for A350, Dab, and A488 or A568, respectively. Density functional theory showed the highest occupied molecular orbital energy and lowest unoccupied molecular orbital energy to be approximately antisymmetric with respect to the geometric center of the ring for A350 (54) and Dabcyl or fluorescein (55), justifying the choice of the dipole center position.…”

Section: Coarse-grained Molecular Dynamicsmentioning

confidence: 97%

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

McCarthy¹,

Savich

Cornea³

et al. 2020

Biophysical Journal

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Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca 2þ , the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca 2þ , the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca 2þ , the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca 2þ stabilized the closed conformation by a factor of two. We conclude that the Ca 2þdependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca 2þ -dependent structural dynamics of bound CaM.

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DFT study of the interaction between the conjugated fluorescein and dabcyl system, using fluorescene quenching method

Cited by 10 publications

References 18 publications

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers

Cell-Penetrating Dabcyl-Containing Tetraarginines with Backbone Aromatics as Uptake Enhancers

PNA-Induced Assembly of Fluorescent Proteins Using DNA as a Framework

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

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