Nanomolar affinity protein trans-splicing monitored in real-time by fluorophore–quencher pairs

2017 ◽  
Vol 53 (3) ◽  
pp. 545-548 ◽  
Author(s):  
M. Braner ◽  
R. Wieneke ◽  
R. Tampé
Keyword(s):  
Real Time ◽  
Protein Labeling ◽  
Online Detection ◽  
Physiological Conditions ◽  
High Affinity ◽  
Cellular Environment ◽  
Trans Splicing

We combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal ‘traceless’ protein labeling at nanomolar concentrations under physiological conditions in cellular environment.

scholarly journals ‘Traceless’ tracing of proteins – high-affinity trans-splicing directed by a minimal interaction pair

2016 ◽  
Vol 7 (4) ◽  
pp. 2646-2652 ◽  
Author(s):  
M. Braner ◽  
A. Kollmannsperger ◽  
R. Wieneke ◽  
R. Tampé
Keyword(s):  
Mammalian Cells ◽  
Protein Labeling ◽  
Terminal Protein ◽  
Site Specific ◽  
High Affinity ◽  
Trans Splicing

Using a minimal lock-and-key element the affinity between the intein fragments for N-terminal protein trans-splicing was significantly increased, allowing for site-specific, ‘traceless’ covalent protein labeling in living mammalian cells at nanomolar probe concentrations.

scholarly journals Systematic Review on Human Skin-Compatible Wearable Photoplethysmography Sensors

2021 ◽  
Vol 11 (5) ◽  
pp. 2313
Author(s):  
Inho Lee ◽  
Nakkyun Park ◽  
Hanbee Lee ◽  
Chuljin Hwang ◽  
Joo Hee Kim ◽  
...  
Keyword(s):  
Systematic Review ◽  
Health Care ◽  
Real Time ◽  
Human Skin ◽  
State Of The Art ◽  
Electronic Devices ◽  
Semiconducting Materials ◽  
Physiological Conditions ◽  
Technological Developments ◽  
Monitoring Technologies

The rapid advances in human-friendly and wearable photoplethysmography (PPG) sensors have facilitated the continuous and real-time monitoring of physiological conditions, enabling self-health care without being restricted by location. In this paper, we focus on state-of-the-art skin-compatible PPG sensors and strategies to obtain accurate and stable sensing of biological signals adhered to human skin along with light-absorbing semiconducting materials that are classified as silicone, inorganic, and organic absorbers. The challenges of skin-compatible PPG-based monitoring technologies and their further improvements are also discussed. We expect that such technological developments will accelerate accurate diagnostic evaluation with the aid of the biomedical electronic devices.

scholarly journals Transmembrane-truncated αvβ3 integrin retains high affinity for ligand binding: evidence for an ‘inside-out’ suppressor?

1998 ◽  
Vol 330 (2) ◽  
pp. 861-869 ◽  
Author(s):  
J. Raj MEHTA ◽  
Beate DIEFENBACH ◽  
Alex BROWN ◽  
Eilish CULLEN ◽  
Alfred JONCZYK ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Αvβ3 Integrin ◽  
Cytoplasmic Domains ◽  
Bivalent Cation ◽  
High Affinity ◽  
Cellular Environment

The molecular mechanisms of αvβ3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human αvβ3 (r-αvβ3) and compared the activation state of these with αvβ3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-αvβ3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental αvβ3 and the receptor in situ on the cell surface. r-αvβ3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-αvβ3. r-αvβ3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native αvβ3. On M21-L4 melanoma cells, αvβ3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated αIIbβ3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified αvβ3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of αIIbβ3 in situ, intracellular controls lower the affinity of αvβ3, and the cytoplasmic domains may act as a target for negative regulators of αvβ3 activity.

Monitoring protein ubiquitination and SUMOylation in real-time by NMR

2020 ◽  
Vol 56 (49) ◽  
pp. 6735-6738
Author(s):  
Batul Ismail Habibullah ◽  
Vasvi Tripathi ◽  
Parag Surana ◽  
Ranabir Das
Keyword(s):  
Real Time ◽  
Physiological Conditions ◽  
Protein Ubiquitination

A new tag-free method detects ubiquitination and SUMOylation of proteins in real time by NMR under physiological conditions.

scholarly journals Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex

2012 ◽  
Vol 443 (1) ◽  
pp. 307-315 ◽  
Author(s):  
Linda Troeberg ◽  
Barbara Mulloy ◽  
Peter Ghosh ◽  
Meng-Huee Lee ◽  
Gillian Murphy ◽  
...  
Keyword(s):  
Sulfated Polysaccharide ◽  
Pentosan Polysulfate ◽  
Endogenous Inhibitor ◽  
Binding Ability ◽  
Physiological Conditions ◽  
Interaction Sites ◽  
High Affinity ◽  
Multiple Protein ◽  
Trimolecular Complex ◽  
Protein Interaction Sites

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX

2007 ◽  
Vol 292 (6) ◽  
pp. C2204-C2212 ◽  
Author(s):  
Johan W. Jonker ◽  
Sandra Musters ◽  
Maria L. H. Vlaming ◽  
Torsten Plösch ◽  
Karin E. R. Gooijert ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Resistance Protein ◽  
Protoporphyrin Ix ◽  
Harderian Gland ◽  
Cancer Resistance ◽  
Erythropoietic Protoporphyria ◽  
Physiological Conditions ◽  
High Affinity ◽  
Expression Site ◽  
Resistance Protein

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria. This protoporphyria is mild compared with ferrochelatase-dependent EPP, and in itself not sufficient to cause phototoxicity, but it might exacerbate the consequences of other porphyrias. In this study, we identified the mouse harderian gland as a novel expression site of Bcrp1. Because of its pronounced role in porphyrin secretion, the harderian gland presents a useful tool to study the mechanism of Bcrp1-related protoporphyria and transport of porphyrins. Bcrp1−/− harderian gland displayed a highly increased accumulation of PPIX glycoconjugates, and a similar shift was seen in Bcrp1−/− liver. Tear- and hepatobiliary excretion data suggest that Bcrp1 controls intracellular levels of PPIX by mediating high affinity transport of its glycoconjugates and possibly low-affinity transport of unconjugated PPIX. This mechanism may allow cells to prevent or reduce cytotoxicity of PPIX under excess conditions, without spillage under physiological conditions where PPIX is needed.

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