scholarly journals Protein conformation: through a lens, darkly

2003 ◽  
Vol 372 (1) ◽  
pp. e1-e2
Author(s):  
Robert INSALL
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Protein Conformation ◽  
Fluorescent Proteins ◽  
Protein Kinase B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Protein ◽  
Insight Into ◽  
Study Offer

This commentary describes the study by Calleja and co-workers, who have used fluorescence resonance energy transfer between two fluorescent proteins fused to one protein kinase (protein kinase B/Akt) to reveal conformational changes in a functional protein. The findings of their study offer an intriguing insight into the behaviour of an enzyme that is essential for multiple aspects of mammalian signalling.

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Development of an optimized backbone of FRET biosensors for kinases and GTPases

2011 ◽  
Vol 22 (23) ◽  
pp. 4647-4656 ◽  
Author(s):  
Naoki Komatsu ◽  
Kazuhiro Aoki ◽  
Masashi Yamada ◽  
Hiroko Yukinaga ◽  
Yoshihisa Fujita ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Fluorescent Proteins ◽  
Rapid Development ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
User Friendliness ◽  
Highly Sensitive ◽  
Fret Biosensors ◽  
Insight Into

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.

Use of a-SiC:H multilayer transducers for detection of fluorescence signals from reactive cyan and yellow fluorophores

2011 ◽  
Vol 1321 ◽  
Author(s):  
P. Louro ◽  
M. Vieira ◽  
M. A. Vieira ◽  
J. Costa ◽  
M. Fernandes ◽  
...  
Keyword(s):  
Spectral Response ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Filters ◽  
Electrical Model ◽  
Excitation Light ◽  
Independent Test ◽  
Opposite Behavior ◽  
Blue Background ◽  
Insight Into

ABSTRACTThe transducer consists of a p-i’(a-SiC:H)-n/p-i(a-Si:H)-n heterostructures produced by PECVD and optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet(400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. The thickness and the absorption coefficient of the i’- and i- layers were tailored for cyan and yellow optical confinement, respectively in the front and back photodiodes acting both as optical filters. The devices were characterized through transmittance and spectral response measurements and under different electrical.To simulate the FRET pairs and the excitation light a chromatic time dependent combination of violet, cyan and yellow wavelengths was applied to the device. The generated photocurrent was measured under negative and positive bias to readout the combined spectra. The independent test signals were chosen in order to sample all the possible chromatic. Different wavelength backgrounds were also superimposed.Results show that under negative bias the phorocurrent signal presents eight separate levels each one assigned to the different polychromatic mixtures. If a blue background is superimposed the yellow channel is enhanced and the cyan suppressed while under red irradiation the opposite behavior occurs. So under appropriated steady state optical bias the sensor will detect separately the cyan and yellow fluorescence pairs. An electrical model, supported by a numerical simulation, gives insight into the transduction mechanism.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals Class Frizzled GPCRs (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Elisa Arthofer ◽  
Jacomijn Dijksterhuis ◽  
Belma Hot ◽  
Paweł Kozielewicz ◽  
Matthias Lauth ◽  
...  
Keyword(s):  
G Proteins ◽  
Conformational Changes ◽  
Hedgehog Signaling ◽  
Cell Signalling ◽  
Resonance Energy Transfer ◽  
Rho Kinase ◽  
Density Lipoprotein ◽  
Resonance Energy ◽  
Scaffolding Protein ◽  
Heterotrimeric G Proteins

Receptors of the Class Frizzled (FZD, nomenclature as agreed by the NC-IUPHAR subcommittee on the Class Frizzled GPCRs [156]), are GPCRs originally identified in Drosophila [17], which are highly conserved across species. While SMO shows structural resemblance to the 10 FZDs, it is functionally separated as it mediates effects in the Hedgehog signaling pathway [156]. FZDs are activated by WNTs, which are cysteine-rich lipoglycoproteins with fundamental functions in ontogeny and tissue homeostasis. FZD signalling was initially divided into two pathways, being either dependent on the accumulation of the transcription regulator β-catenin or being β-catenin-independent (often referred to as canonical vs. non-canonical WNT/FZD signalling, respectively). WNT stimulation of FZDs can, in cooperation with the low density lipoprotein receptors LRP5 (O75197) and LRP6 (O75581), lead to the inhibition of a constitutively active destruction complex, which results in the accumulation of β-catenin and subsequently its translocation to the nucleus. β-Catenin, in turn, modifies gene transcription by interacting with TCF/LEF transcription factors. β-Catenin-independent FZD signalling is far more complex with regard to the diversity of the activated pathways. WNT/FZD signalling can lead to the activation of heterotrimeric G proteins [28, 159, 135], the elevation of intracellular calcium [164], activation of cGMP-specific PDE6 [2] and elevation of cAMP as well as RAC-1, JNK, Rho and Rho kinase signalling [48]. Novel resonance energy transfer-based tools have allowed the study of the GPCR-like nature of FZDs in greater detail. Upon ligand stimulation, FZDs undergo conformational changes and signal via heterotrimeric G proteins [213, 214]. Furthermore, the phosphoprotein Dishevelled constitutes a key player in WNT/FZD signalling. Importantly, FZDs exist in at least two distinct conformational states that regulate the pathway selection [214]. As with other GPCRs, members of the Frizzled family are functionally dependent on the arrestin scaffolding protein for internalization [19], as well as for β-catenin-dependent [12] and -independent [80, 13] signalling. The pattern of cell signalling is complicated by the presence of additional ligands, which can enhance or inhibit FZD signalling (secreted Frizzled-related proteins (sFRP), Wnt-inhibitory factor (WIF), sclerostin or Dickkopf (DKK)), as well as modulatory (co)-receptors with Ryk, ROR1, ROR2 and Kremen, which may also function as independent signalling proteins.

scholarly journals Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Abhishek Mazumder ◽  
Richard H Ebright ◽  
Achillefs Kapanidis
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Extensive Study ◽  
Active Centre ◽  
Transient Nature ◽  
Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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.

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