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•Functionalized cationic gemini (C12-E2O2-C12) and single-chain surfactant (DTAC) instigated the myoglobin unfolding.•Beta-cyclodextrin (β-CD) induced the refolding of the unfolded Mb ...via formation of inclusion complex.•Fluorescence and UV–vis studies unveil nature and extent of binding between cationics and Mb.•Refolding of unfolded Mb using β-CD is found to be a reversible process.
Herein, by pursuing consilience among several methodologies (different spectroscopic, molecular docking, and density functional theory (DFT)), we report that surfactants (functionalized cationic gemini, C12-E2O2-C12, and single-chain surfactant, DTAC) instigated myoglobin (Mb) unfolding and subsequently beta-cyclodextrin (β-CD) induced refolding of this unfolded Mb via inclusion complex formation. Fluorescence and UV–vis results unveil weak binding of the cationics at low concentrations followed by stronger interaction at higher concentrations before reaching the saturation point. Furthermore, refolding of the unfolded Mb using β-CD appears to be a reversible process. However, Mb does not completely recover its structure on refolding. From far-UV circular dichroism (CD) analysis, it is apparent that the α-helical content of Mb decreases/increases during unfolding/refolding process, which is further corroborated by Fourier transform infrared spectroscopy (FT-IR) results. Furthermore, fluctuations in the tertiary structure of Mb were detected through near-UV CD. Synchronous fluorescence together with molecular docking unravels the greater contribution of Trp in the involved interactions. DFT also substantiated the abstraction of C12-E2O2-C12/DTAC by β-CD. Fluorescence, far-UV CD and docking confirm the existence of negligible interaction between Mb and β-CD. The susceptibility of unfolding/refolding of Mb is considerably higher in the case of gemini than DTAC. In light of these results, it is strongly suggested that this work may prove fruitful for protein renaturation studies, which may address the interrelated issues of the biotechnological sector for the production of effective/affordable folding aids that could also be used to treat diseases linked to genetically engineered cells, caused by protein misfolding/aggregation.
Unfolding environmental γ flux spectrum with portable CZT detector Liu, Taiyuan; Xue, Mingxuan; Peng, Haiping ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
June 2024, 2024-06-00, Letnik:
1063
Journal Article
Recenzirano
Environmental γ-rays constitute a crucial source of background in various nuclear, particle and quantum physics experiments. To evaluate the flux rate and the spectrum of γ background, we have ...developed a novel and straightforward approach to reconstruct the environmental γ flux spectrum by applying a portable CZT γ detector and iterative Bayesian unfolding, which possesses excellent transferability for broader applications. In this paper, the calibration and GEANT4 Monte-Carlo modeling of the CZT detector, the unfolding procedure as well as the uncertainty estimation are demonstrated in detail. The reconstructed spectrum reveals an environmental γ flux intensity of 3.3±0.9×107 (m2⋅sr⋅hour)−1 ranging from 73 to 3033 keV, along with characteristic peaks primarily arising from 232Th series, 238U series and 40K. We also give an instance of background rate evaluation with the unfolded spectrum for validation of the approach.
To elucidate the roles of interfacial protein microstructure in regulating techno-functional attributes of complex microgel, this work explored the effects of transglutaminase cross-linking alone or ...in combination with ultrasonication on conformation and functional properties of whey protein isolate-chitooligosaccharide microgel dispersion (WPI–COS). It was found that transglutaminase-crosslinked WPI-COS microgels presented improved interfacial properties when a moderate unfolding process was induced by sonication, validating the existence of optimum flexibility of complex Pickering particles during unfolding/refolding of interfacial proteins. Particularly, TG induced excessive crosslinking of WPI-COS to form aggregates, causing a lower surface hydrophobicity. In contrast, ultrasound was inclined to increase the surface hydrophobicity for TG-catalyzed and uncatalyzed samples, suggesting differential unfolding degrees of WPI induced by transglutaminase and ultrasonication. Correspondingly, interfacial properties of ultrasonicated WPI-COS complexes, as revealed by percentage of adsorbed protein, interfacial tension and three-phase contact angle and emulsifying properties, were improved significantly. Confocal laser scanning microscopy and transmission electron microscopy consistently observed WPI-COS tightly adsorbed at the oil-water interface, with smaller emulsion droplets, more uniform distribution and thicker interfacial films at the oil-water interface in WPI-COS microgel particles with sonication-induced unfolding. This investigation demonstrated the feasibility of conformational regulation of interfacial proteins in tuning techno-functional features of microgel particles.
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•Transglutaminase (TG) coupled to sonication was used to fabricate microgel particle.•TG catalyzed excessive crosslinking of WPI-COS to form aggregates.•Sonication-induced unfolding enhanced the flexibility of microgel particles.•Ultrasound-induced unfolding improved interfacial properties of WPI-COS microgels.•Moderate unfolding counteracted adverse effects of TG-induced WPI aggregation.
The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the ...proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.
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•The Cdc48 ATPase complex binds the ubiquitin chain rather than the attached substrate•One ubiquitin molecule is unfolded by binding to Cdc48 and its Ufd1/Npl4 cofactor•Cdc48 only unfolds polypeptides C-terminal to the unfolded ubiquitin molecule•Unfolded substrate is released from the Cdc48 complex without deubiquitination
The Cdc48 ATPase (p97 or VCP in mammals) complex extracts polyubiquitinated proteins from membranes or macromolecular complexes for proteasomal degradation. Ji et al. clarify the mechanism of substrate processing by showing how the ATPase unfolds one ubiquitin molecule, how it translocates polypeptides with branchpoints, and how it releases its substrates.
Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant ...timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (β/α)8 barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.
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•Kinetic stability and unfolding cooperativity are linked to thermal flexibility•Thermal flexibility is given by residue displacements due to a temperature gradient•There are concerted effects among residues within a certain global spatial context•Protein kinetic stability can be enhanced modifying its thermal flexibility
Quezada et al. propose a quantitative measure of protein flexibility using a residue displacement analysis of temperature-induced unfolding molecular dynamics simulations. Experimentally unfolding activation energies and cooperativities correlate with thermal flexibility. This correlation increases our knowledge on the molecular basis of protein kinetic stability.
We give a comprehensive presentation of the periodic unfolding method for perforated domains, both when the unit hole is a compact subset of the open unit cell and when this is impossible to achieve. ...In order to apply the method to boundary-value problems with nonhomogeneous Neumann conditions on the boundaries of the holes, the properties of the boundary unfolding operator are also extensively studied. The paper concludes with applications to such problems and examples of reiterated unfolding.
The transduction of biological signals often involves structural rearrangements of proteins in response to input signals, which leads to functional outputs. This review discusses the role of ...regulated partial and complete protein unfolding as a mechanism of controlling protein function and the prevalence of this regulatory mechanism in signal transduction pathways. The principles of regulated unfolding, the stimuli that trigger unfolding, and the coupling of unfolding with other well characterized regulatory mechanism are discussed.
Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic ...SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.
Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in the cell, manages misfolded ...proteins to maintain proteostasis is still poorly understood. Here we present a strategy for targeted protein unfolding at the Golgi that enables studying Golgi-related protein quality control and stress-signaling pathways. Targeted protein unfolding is induced by small molecule-based chemical biology approaches-hydrophobic tagging and the use of a destabilization domain. Imaging studies allow visualizing quality control (QC) phenotypes, such as the formation of QC carriers and Golgi-to-endoplasmic reticulum trafficking, and correlating these phenotypes with other trafficking processes.
AAA+ proteolytic machines use energy from ATP hydrolysis to degrade damaged, misfolded, or unneeded proteins. Protein degradation occurs within a barrel-shaped self-compartmentalized peptidase. ...Before protein substrates can enter this peptidase, they must be unfolded and then translocated through the axial pore of an AAA+ ring hexamer. An unstructured region of the protein substrate is initially engaged in the axial pore, and conformational changes in the ring, powered by ATP hydrolysis, generate a mechanical force that pulls on and denatures the substrate. The same conformational changes in the hexameric ring then mediate mechanical translocation of the unfolded polypeptide into the peptidase chamber. For the bacterial ClpXP and ClpAP AAA+ proteases, the mechanical activities of protein unfolding and translocation have been directly visualized by single-molecule optical trapping. These studies in combination with structural and biochemical experiments illuminate many principles that underlie this universal mechanism of ATP-fueled protein unfolding and subsequent destruction.
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