scholarly journals Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ishita Sengupta ◽  
Jayant Udgaonkar
Keyword(s):  
Prion Protein ◽  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monitoring Site ◽  
Site Specific ◽  
Experimental Strategy ◽  
Β Sheets ◽  
Kinetics Of

During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

Calmodulin conformational changes in the activation of protein kinases

2002 ◽  
Vol 30 (2) ◽  
pp. 55-61 ◽  
Author(s):  
K. Török
Keyword(s):  
Energy Transfer ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Process ◽  
Dependent Protein ◽  
Peptide Complexes ◽  
Muscle Myosin ◽  
Kinetics Of

The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/ calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s−1). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and αcCaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of αCaMKII, ATP binding and Thr286 auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to αCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/ calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.

scholarly journals Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function

2020 ◽  
Vol 117 (14) ◽  
pp. 7814-7823 ◽  
Author(s):  
Si Wu ◽  
Liu Hong ◽  
Yuqing Wang ◽  
Jieqiong Yu ◽  
Jie Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Kinetic Measurements ◽  
Kinetics Of ◽  
Conformational Distribution

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

Ultra-sensitive detection of prion protein with a long range resonance energy transfer strategy

2010 ◽  
Vol 46 (43) ◽  
pp. 8285 ◽  
Author(s):  
Ping Ping Hu ◽  
Li Qiang Chen ◽  
Chun Liu ◽  
Shu Jun Zhen ◽  
Sai Jin Xiao ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Prion Protein ◽  
Long Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sensitive Detection

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 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.

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