scholarly journals Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B.

2004 ◽  
Vol 51 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Krystyna Stachowiak ◽  
Monika Tokmina ◽  
Anna Karpińska ◽  
Renata Sosnowska ◽  
Wiesław Wiczk
Keyword(s):  
Amino Acid ◽  
Cysteine Protease ◽  
Cathepsin B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinetic Studies ◽  
Amino Acid Sequences ◽  
Peptide Substrates ◽  
Donor And Acceptor ◽  
Specificity Constant

Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.

scholarly journals Role of Chiral Configuration in the Photoinduced Interaction of D- and L-Tryptophan with Optical Isomers of Ketoprofen in Linked Systems

2021 ◽  
Vol 22 (12) ◽  
pp. 6198
Author(s):  
Aleksandra A. Ageeva ◽  
Ilya M. Magin ◽  
Alexander B. Doktorov ◽  
Victor F. Plyusnin ◽  
Polina S. Kuznetsova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Excited States ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dipole Interaction ◽  
The Body ◽  
Model Systems ◽  
Optical Isomers ◽  
Amino Acid Properties ◽  
Parkinson’S Diseases

The study of the L- and D-amino acid properties in proteins and peptides has attracted considerable attention in recent years, as the replacement of even one L-amino acid by its D-analogue due to aging of the body is resulted in a number of pathological conditions, including Alzheimer’s and Parkinson’s diseases. A recent trend is using short model systems to study the peculiarities of proteins with D-amino acids. In this report, the comparison of the excited states quenching of L- and D-tryptophan (Trp) in a model donor–acceptor dyad with (R)- and (S)-ketoprofen (KP-Trp) was carried out by photochemically induced dynamic nuclear polarization (CIDNP) and fluorescence spectroscopy. Quenching of the Trp excited states, which occurs via two mechanisms: prevailing resonance energy transfer (RET) and electron transfer (ET), indeed demonstrates some peculiarities for all three studied configurations of the dyad: (R,S)-, (S,R)-, and (S,S)-. Thus, the ET efficiency is identical for (S,R)- and (R,S)-enantiomers, while RET differs by 1.6 times. For (S,S)-, the CIDNP coefficient is almost an order of magnitude greater than for (R,S)- and (S,R)-. To understand the source of this difference, hyperpolarization of (S,S)-and (R,S)- has been calculated using theory involving the electron dipole–dipole interaction in the secular equation.

scholarly journals Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

1998 ◽  
Vol 142 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A.K. Kenworthy ◽  
M. Edidin
Keyword(s):  
Energy Transfer ◽  
Lipid Rafts ◽  
Fluorescence Resonance Energy Transfer ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

scholarly journals Development of FRET-based indicators for visualizing homophilic trans interaction of a clustered protocadherin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takashi Kanadome ◽  
Natsumi Hoshino ◽  
Takeharu Nagai ◽  
Tomoki Matsuda ◽  
Takeshi Yagi
Keyword(s):  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Visualization ◽  
Binding Properties ◽  
New Approach ◽  
Self Recognition ◽  
Highly Sensitive ◽  
Donor And Acceptor ◽  
Clustered Protocadherins

AbstractClustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, direct visualization of Pcdh trans interactions across cells remains challenging. Here, we present Förster resonance energy transfer (FRET)-based indicators for directly visualizing Pcdh trans interactions. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. They enabled successful visualization of specific trans interactions of Pcdh and revealed that the Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes.

scholarly journals Fluorescent nucleobase analogues for base–base FRET in nucleic acids: synthesis, photophysics and applications

2018 ◽  
Vol 14 ◽  
pp. 114-129 ◽  
Author(s):  
Mattias Bood ◽  
Sangamesh Sarangamath ◽  
Moa S Wranne ◽  
Morten Grøtli ◽  
L Marcus Wilhelmsson
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Orientation Factor ◽  
Design Synthesis ◽  
Structure And Dynamics ◽  
Donor And Acceptor ◽  
Structure Investigations ◽  
Fret Pair

Förster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base–base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base–base FRET pair, tCO–tCnitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1–qAnitro, increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ 2, have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base–base FRET and that are described here. Base–base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications.

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Kerstin Kühn-Wache ◽  
Joachim W. Bär ◽  
Torsten Hoffmann ◽  
Raik Wolf ◽  
Jens-Ulrich Rahfeld ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Specific Activity ◽  
Selective Inhibition ◽  
Dipeptidyl Peptidase ◽  
Peptide Substrates ◽  
Catalytic Center ◽  
Dipeptidyl Peptidase 4 ◽  
Peptide Family ◽  
Specificity Constant

Abstract Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k cat/K m strongly depends on the amino acid in P1-position for proline, alanine, glycine and serine with 5.0×105 m -1s-1, 1.8×104 m -1s-1, 3.6×102 m -1s-1, 1.1×102 m -1s-1, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P1-position is the same range as the k cat/K m values of NPY derivatives containing alanine or serine in P1-position with 4×105 m -1s-1, 9.5×105 m -1s-1 and 2.1×105 m -1s-1, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This ‘secondary’ binding site interaction depends on the amino acid sequence in P4′–P8′-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters

Nanoscale ◽  
2016 ◽  
Vol 8 (26) ◽  
pp. 13006-13016 ◽  
Author(s):  
Roopali Prajapati ◽  
Surajit Chatterjee ◽  
Krishna K. Kannaujiya ◽  
Tushar Kanti Mukherjee
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Silver Nanoclusters ◽  
Donor And Acceptor

scholarly journals Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery

2014 ◽  
Vol 20 (4) ◽  
pp. 508-518 ◽  
Author(s):  
Christine J. Rossant ◽  
Carl Matthews ◽  
Frances Neal ◽  
Caroline Colley ◽  
Matthew J. Gardener ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Drug Discovery ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Donor And Acceptor ◽  
Wide Range ◽  
Fluorescence Resonance

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

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