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Background and objectives: The cyclic adenosine monophosphate Receptor Protein of Mycobacterium tuberculosis (CRPMt) (Rv3676), is a global transcriptional regulator and plays a pivotal role in the survival and infection of Mycobacterium. This signaling protein (CRPMt) shares several common structural and functional features with the CRP from Escherichia coli. Structurally, CRPMt is a homodimer that undergoes allosteric changes upon cyclic AMP binding. This binding also triggers the activation of several genes responsible for various physiological processes in this bacterium. Despite the importance of CRP for mycobacterial survival, limited information is available regarding the stability and unfolding properties of the protein. The main objective of this study is to study stability, unfolding and dynamics of CRPMt in terms of its structure. Methods: In this study, we monitored the stability and unfolding of CRPMt using various biophysical and computational techniques. Results: We experimentally studied protein unfolding in the presence of chemical denaturants [urea and guanidine hydrochloride (GdnHCl)]. The results from these chemical-induced unfolding studies suggest that CRPMt follows a two-state transition and that chemical-induced protein denaturation is reversible. According to circular dichroism and activity data, CRPMt structure and function were restored upon refolding. We also studied the stability and unfolding of the CRPMt protein against temperature variations and protease action (trypsin). Limited proteolysis experiments provide insights into the minimum domain structure requirement for CRPMt activity. Interestingly, temperature-induced CRPMt unfolding was completely different compared to chemical-induced unfolding. The thermal unfolding of CRPMt was found to be irreversible, leading to the formation of insoluble aggregates at elevated temperatures. To understand why the thermal unfolding of the protein differed from chemically induced unfolding, we carried out a detailed molecular dynamics simulation analysis of the protein at three different temperatures. The results from these molecular dynamics simulations mechanistically validate the significant differences between chemical and temperature-induced CRPMt unfolding. Conclusion: Our study provides detailed insights into the stability and folding/unfolding properties of CRPMt, which could be useful in developing new anti-mycobacterial medicines. [ABSTRACT FROM AUTHOR]

α‐Cyanostilbene represents one of the easily functionalized aggregation‐induced emission (AIE) scaffolds. It has been widely adopted for the construction of fluorescent materials for broad applications. Here, we further expanded the utilization of α‐cyanostilbene derivatives for the detection of hypoxia or proteostasis imbalance in live cells. Four different amine containing donors were introduced to construct α‐cyanostilbene derivatives (R‐ASC) with donor‐acceptor scaffolds. Equipped with the cysteine (Cys) reactive group, maleimide (MI), R‐ASC‐MI shows fluorescence turn‐on property upon binding with unfolded proteins in vitro and in live cells under proteostatic stress. By virtue of R‐ASC‐MI, the level of unfolded protein loads in cells can be quantified by flow cytometry, or visualized under microscope. Furthermore, we also characterized the performance of R‐ASC‐NO2, synthetic precursors of R‐ASC‐MI, in cellular hypoxia. R‐ASC‐NO2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN3) induced or atmospheric (1 % O2) hypoxia. Together, the advantages of easy modification and high signal‐to‐noise ratio of new α‐cyanostilbene derivatives reported in this work highlight the great potential of α‐cyanostilbene in constructing functional biosensors and many other domains. [ABSTRACT FROM AUTHOR]

Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape. [ABSTRACT FROM AUTHOR]

AbstractEven after the centenary celebration of insulin discovery, there prevail challenges concerning insulin aggregation, not only after repeated administration but also during industrial production, storage, transport, and delivery, significantly impacting protein quality, efficacy, and effectiveness. The aggregation reduces insulin bioavailability, increasing the risk of heightened immunogenicity, posing a threat to patient health, and creating a dent in the golden success story of insulin therapy. Insulin experiences various physicochemical and mechanical stresses due to modulations in pH, temperature, ionic strength, agitation, shear, and surface chemistry, during the upstream and downstream bioprocessing, resulting in insulin unfolding and subsequent fibrillation. This has fueled research in the pharmaceutical industry and academia to unveil the mechanistic insights of insulin aggregation in an attempt to devise rational strategies to regulate this unwanted phenomenon. The present review briefly describes the impacts of environmental factors of bioprocessing on the stability of insulin and correlates with various intermolecular interactions, particularly hydrophobic and electrostatic forces. The aggregation-prone regions of insulin are identified and interrelated with biophysical changes during stress conditions. The quest for novel additives, surface-active agents, and bioderived peptides in decelerating insulin aggregation, which results in overall structural stability, is described. We hope this review will help tackle the real-world challenges of insulin aggregation encountered during bioprocessing, ensuring safer, stable, and globally accessible insulin for efficient management of diabetes. [ABSTRACT FROM AUTHOR]

The Arrhenius energy of activation of unfolding Ea unfolding and Gibbs free energy of unfolding ΔG unfolding have been calculated utilizing DSC differential scanning calorimetry for 4 mAbs (1 biosimilar) in 3 formulations. DSC derived ΔTm melting temperature changes for each mAb domain (CH2, Fab, CH3) at calorimetric scan rates at 60 °C, 90 °C, 150 °C and 200 °C / hr. were utilized to calculate the kinetic Ea unfolding. The DSC derived Ea trend with observed aggregate formation and can be used to predict%HMW formation post 9-month storage at 5 °C and 40 °C for all formulations analyzed. Additionally, thermodynamic ΔG unfolding energies were also derived (Tm, ΔCp and ΔH measurements) for each mAb at every scan rate to observe scan rate dependence of ΔG and for extrapolation to 0 °C/hr. (to report ΔG at true equilibrium conditions). Both derived thermodynamic ΔG and kinetic Ea energies were combined to build full energetic landscapes for mAb unfolding and aggregation. Statistical multivariate analysis of kinetic (Ea CH2, Ea Fab, Ea CH3) energies, thermodynamic (ΔG 5 ° C and ΔG 40 ° C) energies and in-silico modeled surface properties was also performed. Analysis revealed key significant parameters contributing to aggregation. These parameters were utilized to build predictive aggregation models for 25 mg/mL mAb formulations stored 9-months at 5 °C and 40 °C. [ABSTRACT FROM AUTHOR]

In eukaryotes, the ubiquitin‐proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore‐1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N‐terminus, remain unknown. We determined how the location of glycine‐rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine‐rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding. [ABSTRACT FROM AUTHOR]

Drug-induced cardiotoxicity (DICT) poses a significant challenge in the prognosis of cancer patients, particularly with the use of antineoplastic agents like anthracyclines and targeted therapies such as trastuzumab. This review delves into the intricate interplay between drugs and proteins within cardiac cells, focusing on the role of proteostasis as a therapeutic target for mitigating cardiotoxicity. We explore the in vivo modeling of proteostasis, highlighting the complex intracellular environment and the emerging techniques for monitoring proteostasis. Additionally, we discuss how cardiotoxic drugs disrupt protein homeostasis through direct chemical denaturation, endoplasmic reticulum stress, unfolded protein response, chaperone dysfunction, impairment of the proteasome system, and dysregulation of autophagy. Finally, we provide insights into the applications of cardioprotective drugs targeting proteostasis to prevent cardiotoxicity and the adoption of structural proteomics to evaluate potential cardiotoxicity. By gaining a deeper understanding of the role of proteostasis underlying DICT, we can pave the way for the development of targeted therapeutic strategies to safeguard cardiac function while maximizing the therapeutic potential of antineoplastic drugs.

The experiments involving protein denaturation and refolding serve as the foundation for predicting the three-dimensional spatial structures of proteins based on their amino acid sequences. Despite significant progress in protein structure engineering, exemplified by AlphaFold2 and OmegaFold, there remains a gap in understanding the folding pathways of polypeptide chains leading to their final structures. We developed a lightweight tool for protein unfolding visualization called PUV whose graphics design is mainly implemented by OpenGL. PUV leverages principles from molecular biology and physics, and achieves rapid visual dynamics simulation of protein polypeptide chain unfolding through mechanical force and atom-level collision detection and elimination. After a series of experimental validations, we believe that this method can provide essential support for investigating protein folding mechanisms and pathways.

This study assessed the effect of whey protein isolate (WPI) and its coconut oil complexes (CO-WPI) on the viability of Limosilactobacillus reuteri KUB-AC5 during spray drying and storage. Varied CO concentrations (0%, 4%, and 8%, w/w) and spray drying temperatures (110 °C and 130 °C) influenced secondary structures, free sulfhydryl groups, disulfide bonds, and DPPH• and ABTS•+ scavenging activities of WPI. Throughout the spray drying and storage processes, CO-WPI effectively reduced lipid peroxidation in the probiotic membranes, as detected by advanced 1H-nuclear magnetic resonance, compared to WPI alone. Microencapsulation in 4CO-WPI at 110 °C provided the highest cell viability during drying (1.34 × 1011 CFU g−1), coupled with the highest cholesterol assimilation (58.43%). After 12 months at 4 °C, viability remained high (5.57 × 1010 CFU g−1). This study demonstrated CO-WPI's potential in reducing oxidative damage and improving probiotic survival, highlighting its efficacy as a microencapsulant in spray drying. [ABSTRACT FROM AUTHOR]

Protein sequencing is crucial for understanding the complex mechanisms driving biological functions. However, proteins are usually folded in their native state and the mechanism of fast protein conformation transitions still remains unclear, which make protein sequencing challenging. Molecular dynamics simulations with accurate force field are now able to observe the entire folding/unfolding process, providing valuable insights into protein folding mechanisms. Given that proteins can be unfolded, nanopore technology shows great potential for protein sequencing. In this study, we proposed to use MoS2/SnS2 heterostructures to firstly unfold proteins and then detect them by a nanopore in the heterostructural membrane. All-atom molecular dynamics simulations performed in this work provided rich atomic-level information for a comprehensive understanding of protein unfolding process and mechanism on the MoS2/SnS2 heterostructure, it was found that the strong binding of protein to SnS2 nanostripe and hydrogen bond breaking were the main reasons for unfolding the protein on the heterostructure. After the protein was fully unfolded, it was restrained on the nanostripe because of the affinity of protein to the SnS2 nanostripe. Thus by integrating the proposed unfolding technique with nanopore technology, detection of linear unfolded peptide was realized in this work, allowing for the identification of protein components, which is essential for sequencing proteins in the near future. [ABSTRACT FROM AUTHOR]

• Thermal stability of polysorbate-degradative enzymes was assessed with chemical proteomics and biophysical studies. • Brief heat treatment inactivated polysorbate-degradative enzymes and had minimal impact on the therapeutic antibody. • Heat inactivation of protein-A pool decelerated polysorbate degradation. Polysorbate degradation in biotherapeutics formulations is an industry-wide problem and mainly caused by residual host cell-derived enzymes. We present a proof-of-concept study of a control strategy which takes advantage of lower thermal stability of such enzymes relative to therapeutic proteins. We profiled heat sensitivity of host cell–derived enzyme activity with chemical proteomics and observed that PLA2G7 became inactive after brief heating. Further biophysical studies indicated that these enzymes were less thermally stable than a monoclonal antibody. Importantly, brief heat treatment had minimal impact on the stability of the antibody. Consequently, heat inactivation of polysorbate-spiked protein-A pool decelerated polysorbate degradation. This study suggests that heat inactivation of host cell–derived enzymes could be a control stragy for polysorbate degradation. [ABSTRACT FROM AUTHOR]

Withaferin A (WA) and celastrol (CEL) are major bioactive components of plants that have been widely employed in traditional medicine. The pleiotropic activities of plant preparations and the isolated compounds in vitro and in vivo have been documented in hundreds of studies. Both WA and CEL were shown to have anticancer activity. Although WA and CEL belong to different chemical classes, our synthesis of the available information suggests that the compounds share basic mechanisms of action. Both WA and CEL bind covalently to numerous proteins, causing the partial unfolding of some of these proteins and of many bystander proteins. The resulting proteotoxic stress, when excessive, leads to cell death. Both WA and CEL trigger the activation of the unfolded protein response (UPR) which, if the proteotoxic stress persists, results in apoptosis mediated by the PERK/eIF-2/ATF4/CHOP pathway or another UPR-dependent pathway. Other mechanisms of cell death may play contributory or even dominant roles depending on cell type. As shown in a proteomic study with WA, the compounds appear to function largely as electrophilic reactants, indiscriminately modifying reachable nucleophilic amino acid side chains of proteins. However, a remarkable degree of target specificity is imparted by the cellular context. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.

The 26S proteasome is responsible for the unfolding and degradation of intracellular proteins in eukaryotes. A hexameric ring of ATPases (Rpt1-Rpt6) grabs onto substrates and unfolds them by pulling them through a central pore and translocating them into the 20S degradation chamber. A set of pore loops containing a so-called aromatic paddle motif in each Rpt subunit is believed to be important for the proteasome's ability to unfold and translocate substrates. Based on structural and mechanistic experiments, paddles from adjacent Rpt subunits, which are arrayed in a spiral staircase conformation, grip and pull on the substrate in a hand-over-hand type mechanism, disengaging at the bottom of the staircase and re-engaging at the top. We tested the contribution of the aromatic paddles to unfolding substrates of differing stabilities by mutating the paddles singly or in combination. For an easy-to-unfold substrate (a circular permutant of green fluorescent protein; GFP), mutations had little effect on degradation rates. For a substrate with moderate stability (enhanced GFP), there were modest effects of individual mutations on GFP unfolding rates, and alternating aromatic paddle mutants had a larger detrimental effect on unfolding than sequential mutants. For a more stable substrate (superfolder GFP), unfolding is overall slower, and multiple simultaneous mutations essentially prevent unfolding. Our results highlight the context-dependent need for grip during unfolding, support the hand-over-hand model for substrate unfolding and translocation, and suggest that for hard-to-unfold substrates, it is important to have simultaneous strong contacts to the substrate for unfolding to occur. The results also suggest a kinetic proofreading model, where substrates that cannot be easily unfolded are instead clipped, removing the initiation region and preventing futile unfolding attempts. • Proteasomal ATPases contain pore loop paddles that unfold and translocate substrates. • Mutation of pore loop tyrosines impairs unfolding of only difficult to unfold substrates. • Simultaneous paddle-substrate contacts facilitate unfolding of challenging substrates. • If unfolding becomes too slow, an alternative clipping reaction predominates. [ABSTRACT FROM AUTHOR]

The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein–protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein–protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption. [ABSTRACT FROM AUTHOR]

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

BackgroundThe hydrophobic pocket (HP) of HIV-1 glycoprotein-41 ectodomain is defined by two chains of the N-heptad repeat trimer, within the protein-protein interface that mediates 6HB formation. It is a potential target for inhibitors of viral fusion, but its hydrophobic nature and proximity to membrane in situ has precluded ready analysis of inhibitor interactions.MethodsWe evaluated the sensitivity of 19F NMR and fluorescence for detecting peptide and small molecule binding to the HP and explored the effect of non-denaturing detergent or phospholipid as cosolvents and potential mimics of the membrane environment surrounding gp41.ResultsChemical shifts of aromatic fluorines were found to be sensitive to changes in the hydrogen bonding network that occurred when inhibitors transitioned from solvent into the HP or into ordered detergent micelles. Fluorescence intensities and emission maxima of autofluorescent compounds responded to changes in the local environment.ConclusionsGp41 - ligand binding occurred under all conditions, but was diminished in the presence of detergents. NMR and fluorescence studies revealed that dodecylphosphocholine (DPC) was a poor substitute for membrane in this system, while liposomes could mimic the membrane surroundings.General significanceOur findings suggest that development of high potency small molecule binders to the HP may be frustrated by competition between binding to the HP and binding to the bilayer membrane.

Most proteins fold into characteristic three-dimensional structures. The rate of folding and unfolding varies widely and can be affected by variations in proteins. We developed a novel machine-learning-based method for the prediction of the folding rate effects of amino acid substitutions in two-state folding proteins. We collected a data set of experimentally defined folding rates for variants and used them to train a gradient boosting algorithm starting with 1161 features. Two predictors were designed. The three-class classifier had, in blind tests, specificity and sensitivity ranging from 0.324 to 0.419 and from 0.256 to 0.451, respectively. The other tool was a regression predictor that showed a Pearson correlation coefficient of 0.525. The error measures, mean absolute error and mean squared error, were 0.581 and 0.603, respectively. One of the previously presented tools could be used for comparison with the blind test data set, our method called PON-Fold showed superior performance on all used measures. The applicability of the tool was tested by predicting all possible substitutions in a protein domain. Predictions for different conformations of proteins, open and closed forms of a protein kinase, and apo and holo forms of an enzyme indicated that the choice of the structure had a large impact on the outcome. PON-Fold is freely available. [ABSTRACT FROM AUTHOR]

The AAA+‐ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo‐EM structures of p97 in the process of disassembling a protein phosphatase‐1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor‐3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N‐domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N‐domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral‐shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N‐domain and central channel, suggesting a hold‐and‐extract mechanism for p97‐mediated disassembly. Synopsis: It remains unclear how substrate complexes are loaded onto the p97/VCP AAA+‐ATPase and disassembled. Here, structures show p97 bound to a PP1‐regulatory complex undergoing disassembly and suggest a hold‐and‐extract mechanism. Cryo‐EM shows p97 and its p37 adapter loaded with a ternary PP1 complex as a substrate for disassembly.The PP1 partner SDS22 is bound tightly and directly to an N‐domain of p97.The I3 partner of PP1 is inserted into the central channel of p97 while still attached to PP1.p97 is in an active staircase conformation primed to strip the I3 subunit off PP1. [ABSTRACT FROM AUTHOR]

The flexibility of the ATP synthase's β subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated β subunit (Tβ) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tβ shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the β‐sheet residual structure at high temperature. We determined that part of the stability origin of Tβ is due to a transversal hydrophobic array that crosses the β‐barrel formed at the N‐terminal domain and the Rossman fold of the nucleotide‐binding domain (NBD), while the helix bundle of the C‐terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase. [ABSTRACT FROM AUTHOR]

Unfolding of proteins is a crucial event that leads to a range of deleterious results, such as inactivation of a therapeutic protein or onset of a misfolding disease. A better insight onto details of unfolding mechanisms may help to design more stable proteins or prevent/delay the unfolding process by suitable ligands. In this study, we have used molecular dynamics (MD) simulations (AMBER14 force field implemented in YASARA) to investigate the unfolding of an albumin binding domain at atomic level. The aim was to find and target a possible initial site of the peptide's unfolding. The original peptide has a relatively simple structure composed of three alpha helices which were exposed to high temperature. A potential critical point in unfolding (Lys 11) was localized based on secondary structure changes. Eight different mutations with diverse physicochemical properties were then proposed for this site, and the mutated peptides were simulated to check their effect on the unfolding process. We found that replacement of the critical residue with aliphatic ones seems to be better suited for this position in comparison with charged and bulkier aromatic residues. We believe that this approach could be further developed in order to provide a simple and relatively fast tool to rationally stabilize peptides. [ABSTRACT FROM AUTHOR]

Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1-overexpressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD.SIGNIFICANCE STATEMENT In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated whether neuronal overexpression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 overexpression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of α-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop-substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Following assembly, the anthrax protective antigen (PA) forms an oligomeric translocon that unfolds and translocates either its lethal factor (LF) or edema factor (EF) into the host cell. Here, we report the cryo-EM structures of heptameric PA channels with partially unfolded LF and EF at 4.6 and 3.1-Å resolution, respectively. The first α helix and β strand of LF and EF unfold and dock into a deep amphipathic cleft, called the α clamp, which resides at the interface of two PA monomers. The α-clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins. EF undergoes a largescale conformational rearrangement when forming the complex with the channel. A critical loop in the PA binding interface is displaced for about 4 Å, leading to the weakening of the binding interface prior to translocation. These structures provide key insights into the molecular mechanisms of translocation-coupled protein unfolding and translocation.

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aarssti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aarssti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aarssti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aarssti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Exposure of many cancer cells, including multiple myeloma cells, to cytotoxic concentrations of natural products celastrol and withaferin A or synthetic compounds of the IHSF series resulted in denaturation of a luciferase reporter protein. Proteomic analysis of detergent-insoluble extract fractions from HeLa-derived cells revealed that withaferin A, IHSF058 and IHSF115 caused denaturation of 915, 722 and 991 of 5132 detected cellular proteins, respectively, of which 440 were targeted by all three compounds. Western blots showed that important fractions of these proteins, in some cases approaching half of total protein amounts, unfolded. Relatively indiscriminate covalent modification of target proteins was observed; 1178 different proteins were modified by IHSF058. Further illustrating the depth of the induced proteostasis crisis, only 13% of these proteins detectably aggregated, and 79% of the proteins that aggregated were not targets of covalent modification. Numerous proteostasis network components were modified and/or found in aggregates. Proteostasis disruption caused by the study compounds may be more profound than that mediated by proteasome inhibitors. The compounds act by a different mechanism that may be less susceptible to resistance development. Multiple myeloma cells were particularly sensitive to the compounds. Development of an additional proteostasis-disrupting therapy of multiple myeloma is suggested. [ABSTRACT FROM AUTHOR]

We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change Cp(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile Cp(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical–mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical–mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical–mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies. [ABSTRACT FROM AUTHOR]

Withaferin A (WA) and celastrol (CEL) are major bioactive components of plants that have been widely employed in traditional medicine. The pleiotropic activities of plant preparations and the isolated compounds in vitro and in vivo have been documented in hundreds of studies. Both WA and CEL were shown to have anticancer activity. Although WA and CEL belong to different chemical classes, our synthesis of the available information suggests that the compounds share basic mechanisms of action. Both WA and CEL bind covalently to numerous proteins, causing the partial unfolding of some of these proteins and of many bystander proteins. The resulting proteotoxic stress, when excessive, leads to cell death. Both WA and CEL trigger the activation of the unfolded protein response (UPR) which, if the proteotoxic stress persists, results in apoptosis mediated by the PERK/eIF-2/ATF4/CHOP pathway or another UPR-dependent pathway. Other mechanisms of cell death may play contributory or even dominant roles depending on cell type. As shown in a proteomic study with WA, the compounds appear to function largely as electrophilic reactants, indiscriminately modifying reachable nucleophilic amino acid side chains of proteins. However, a remarkable degree of target specificity is imparted by the cellular context.

The UV photodissociation kinetics of tryptophan amino acid, Trp, attached to the membrane of bacteria, Escherichia coli and Bacillus subtilis, have been studied by means of normal and synchronous fluorescence. Our experimental data suggest that the fluorescence intensity of Trp increases during the first minute of irradiation with 250 nm to ∼ 280 nm, 7 mW/cm2 UV light, and subsequently decreases with continuous irradiation. During this short, less than a minute, period of time, 70% of the 107 cell per milliliter bacteria are inactivated. This increase in fluorescence intensity is not observed when tryptophan is in the free state, namely, not attached to a protein, but dissolved in water or saline solution. This increase in fluorescence is attributed to the additional fluorescence of tryptophan molecules formed by protein unfolding, the breakage of the bond that attaches Trp to the bacterial protein membrane, or possibly caused by the irradiation of 2 types of tryptophan residues that photolyze with different quantum yields.

The non-thermal effect of electromagnetic field (NEF) on the structural and thermal properties of heat-resistant globular sunflower protein isolate was investigated by exposing samples to varying power levels (70 W, defrost (DF), and 350 W). Only the lowest consecutive power modes of the electromagnetic unit were conducted to complete at least two exposure cycles (for certainty) without exceeding 45 °C (non-thermal processing condition). The total polar amino acid content of the treated samples decreased by 14% with NEF applications since polar amino acids were the main targets of the electromagnetic field due to absorbing that energy as kinetic energy and inducing structural changes. The DF and 350 W treatments dissipated large particles/aggregates over 5000 nm completely. The treatment with the lowest power (70 W) produced the lowest average particle size (14% decrease) while it increased after the DF and 350 W applications (34 and 16%, respectively), which indicated partial unfolding and/or reaggregation. Less ordered structures had increased α-helix (max with 350 W by 22%) and decreased β-sheet contents (max with 70 W by 30%) after the NEF treatments. The tertiary structures of the samples changed significantly following the NEF treatments with a blue shift on emission maxima with different fluorescence intensities. The thermal stability of the samples was analyzed with DSC and TGA; lower peak temperature (decreased by 28%) and denaturation enthalpy (decreased by 82%) besides higher gravimetric loss (by 1.3%) were obtained for DF and 350 W, compared to the control. The NEF could be considered a promising approach for structural alteration of globular sunflower protein. [ABSTRACT FROM AUTHOR]

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding. [ABSTRACT FROM AUTHOR]

Herceptin (or trastuzumab) is an important therapeutic monoclonal antibody (mAb) used in the treatment of HER2‐positive breast cancer. Real‐time counting and characterization of Herceptin is a fundamental step in the field of disease‐related diagnosis and therapy. Solid‐state nanopore‐based biosensors have been proved to hold great potential in characterizing the properties of proteins at the single‐molecule level for in vitro diagnosis. Here, the label‐free detection and detailed translocation dynamics study of Herceptin using solid‐state nanopores are demonstrated. By constricting nanopore size close to the size of Herceptin, the detection sensitivity and temporal resolution have been significantly improved, allowing the delicate probing of the structural information of single‐molecule Herceptin. Therefore, three types of Herceptin translocation events are identified through nanopores, single‐level, multi‐level and spike‐like events, emerged at different voltages regimes, indicating the unfolding kinetics of Herceptin under electric field. The potential influence of a high electric field on complex biomolecules is highlighted and a novel prospective platform is provided for label‐free detection of single‐molecule therapeutic monoclonal antibodies via solid‐state nanopores as a miniaturized biomedical device. [ABSTRACT FROM AUTHOR]

Unfolding can interrupt the activity of enzymes. Lipase, the enzyme responsible for triglyceride catalysis, can be deactivated by unfolding, which can significantly affect the yield of enzymatic processes in biochemical engineering. Different agents can induce lipase unfolding, among which we study the impact of urea as a strong denaturant. Unfolding weakens the rigidity and stability of globular proteins, thereby changing the viscoelastic properties of the protein adsorbed layers. These changes can be detected and quantified using interfacial dilational rheology. The urea-induced unfolding of lipase destructs its globular structure, making it more flexible. The interfacial tension and viscoelastic moduli of lipase adsorbed layers reduce upon the addition of urea in the range of studied concentrations. The results agree with the theory that, upon unfolding, a distal region of the loop and tail domain forms adjacent to the proximal region of the interface. The exchange of matter between these regions reduces the viscoelasticity of the unfolded lipase adsorbed layers. Additionally, unfolding reduces the rigidity and brittleness of the lipase adsorbed layers: the aged adsorbed layer of native lipase can break upon high-amplitude perturbations of the interfacial area, unlike the case for urea-induced unfolded lipase. [ABSTRACT FROM AUTHOR]

Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native structure to an unfolded protein or vice versa, identifying the intermediates, and defining the role of sequence and structure en route remain outstanding problems in the field. It is even more challenging to capture the events at atomistic resolution. X‐ray diffraction has so far been used to understand how urea interacts with and unfolds two stable globular proteins. Here, we present the case study on PSMD10Gankyrin, a prototype for a moderately stable, non‐globular repeat protein, long and rigid, with its termini located at either end. We define structural changes in the time dimension using low urea concentrations to arrive at the following conclusions. (a) Unfolding is rapidly initiated at the C‐terminus, slowly at the N‐terminus, and proceeds inwards from both ends. (b) C‐terminus undergoes an α to 310 helix transition, representing the structure of a potential early unfolding intermediate before disorder sets in. (c) Distinct and progressive changes in the electrostatic landscape of PSMD10Gankyrin were observed, indicative of conformational changes in the seemingly inflexible motif involved in protein–protein interaction. We believe this unique study will open up the field for better and bolder queries and increase the choice of model proteins for a better understanding of the challenging problems of protein folding, protein interactions, protein degradation, and diseases associated with misfolding. PDB Code(s): 7VXV, 7VXW, 7VY4 and 7VY7; [ABSTRACT FROM AUTHOR]

Fibrinogen (Fg), an essential plasma glycoprotein involved in the coagulation cascade, undergoes structural alterations upon exposure to various chemicals, impacting its functionality and contributing to pathological conditions. This research article explored the effects of 4-Chloro- o -phenylenediamine (4-Cl-o-PD), a common hair dye component (IUPAC = 1-Chloro-3,4-diaminobenzene), on human fibrinogen through comprehensive computational, biophysical, and biochemical approaches. The formation of a stable ligand-protein complex is confirmed through molecular docking and molecular dynamics simulations, revealing possible interaction having a favorable −4.8 kcal/mol binding energy. Biophysical results, including UV–vis and fluorescence spectroscopies, corroborated with the computational findings, whereas Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD) provide insights into the alterations of secondary structures upon interaction with 4-Cl-o-PD. Anilinonaphthalene-sulfonic acid (ANS) fluorescence showed a partially unfolded protein, with enhanced α to β-sheet transition as evidenced by thioflavin T (ThT) spectroscopy and microscopy. Moreover, biochemical assays confirmed the formation of carbonyl compounds that may be responsible for the oxidation of methionine residues in fibrinogen. Electrophoresis and electron microscopy confirmed the formation of aggregates. Our findings elucidate the interaction pattern of 4-Cl-o-PD with Fg, leading to structural perturbation, which may have potential implications for fibrinogen misfolding or its aggregation. Protein aggregation or its misfolded products affect peripheral tissues and the central nervous system. Many chronic progressive diseases, like type II diabetes mellitus, Alzheimer's disease, Parkison's disease, and Creutzfeldt-Jakob disease are associated with intrinsically aberrant disordered proteins. Understanding these interactions may offer new perspectives on the safety and biocompatibility of dye compounds, which may contribute to developing improved strategies for acquired amyloidogenesis. [ABSTRACT FROM AUTHOR]

Whey protein isolates (WPI) were treated with different ethanol level (20, 40, 60, and 80%, v/v) to promote structural unfolding and subsequent aggregation. In general, protein aggregation gradually increased with increasing ethanol level in a dose-dependent manner, which was implied by notably increased turbidity and gradually decreased solubility. The formation of aggregates, which were confirmed by the results of circular dichroism spectrum and total sulfhydryl content, were promoted mainly through disulfide bonds and intra-molecular hydrogen bonds. Moreover, ethanol treated WPI (E-WPI) had significantly enhanced antioxidant activities over native WPI, which was mainly attribute to the higher contents of specific amino acids (such as hydrophobic amino acids, aromatic amino acids, and sulfur-containing amino acids), and E-WPI prepared with moderate ethanol concentration (40% in our present study) exhibited the highest antioxidant activities. These results reveal that antioxidant activities of WPI can be increased by ethanol treatment and are possibly achieved through molecular unfolding of native WPI.

The AAA+ ATPase p97 (also called VCP or Cdc48) is a major protein unfolding machine with hundreds of clients in diverse cellular pathways that are critical for cell homeostasis, proliferation and signaling. In this review, we summarize recent advances in understanding how diverse client proteins are targeted to the p97 machine to facilitate client degradation or to strip clients from binding partners for regulation. We describe an elaborate system that is governed by at least two types of alternative adapters. The Ufd1-Npl4 adapter along with accessory adapters targets ubiquitylated clients in the majority of pathways and uses ubiquitin as a universal unfolding tag. In contrast, the family of SEP-domain adapters such as p37 can target clients directly to p97 in a ubiquitin-independent manner. Despite the different targeting strategies, both pathways converge by inserting the client into the p97 pore to initiate a peptide threading mechanism through the central channel of p97 that drives client protein unfolding, protein extraction from membranes and protein complex disassembly processes.

Inorganic polyphosphate (polyP) constitutes one of the most conserved and ubiquitous molecules in biology. Recent work in bacteria demonstrated that polyP increases oxidative stress resistance by preventing stress-induced protein aggregation and promotes biofilm formation by stimulating functional amyloid formation. To gain insights into these two seemingly contradictory functions of polyP, we investigated the effects of polyP on the folding model lactate dehydrogenase. We discovered that the presence of polyP during the thermal unfolding process stabilizes folding intermediates of lactate dehydrogenase as soluble micro-β-aggregates with amyloid-like properties. Size and heterogeneity of the oligomers formed in this process were dependent on the polyP chain length, with longer chains forming smaller, more homogenous complexes. This ability of polyP to stabilize thermally unfolded proteins even upon exposure to extreme temperatures appears to contribute to the observed resistance of uropathogenic Escherichia coli toward severe heat shock treatment. These results suggest that the working mechanism of polyP is the same for both soluble and amyloidogenic proteins, with the ultimate outcome likely being determined by a combination of polyP chain length and the client protein itself. They help to explain how polyP can simultaneously function as general stress-protective chaperone and instigator of amyloidogenic processes in vivo.

In cyanobacteria, the KaiABC posttranslational oscillator drives circadian rhythms of gene expression and controls the timing of cell division. The Kai-based oscillator can be reconstituted in vitro, demonstrating that the clock can run without protein synthesis and degradation; however, protein degradation is known to be important for clock function in vivo. Here, we report that strains deficient in the ClpXP1P2 protease have, in addition to known long-period circadian rhythms, an exaggerated ability to synchronize with the external environment (reduced "jetlag") compared with WT strains. Deletion of the ClpX chaperone, but not the protease subunits ClpP1 or ClpP2, results in cell division defects in a manner that is dependent on the expression of a dusk-peaking factor. We propose that chaperone activities of ClpX are required to coordinate clock control of cell division whereas the protease activities of the ClpXP1P2 complex are required to maintain appropriate periodicity of the clock and its synchronization with the external environment.

Although helices play key roles in peptide-protein and protein-protein interactions, the helical conformation is generally unstable for short peptides (10-15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a "capped-strapped" peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and Tm ≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.

While it is widely appreciated that the denatured state of a protein is a heterogeneous conformational ensemble, there is still debate over how this ensemble changes with environmental conditions. Here, we use single-molecule chemo-mechanical unfolding, which combines force and urea using the optical tweezers, together with traditional protein unfolding studies to explore how perturbants commonly used to unfold proteins (urea, force, and temperature) affect the denatured-state ensemble. We compare the urea m-values, which report on the change in solvent accessible surface area for unfolding, to probe the denatured state as a function of force, temperature, and urea. We find that while the urea- and force-induced denatured states expose similar amounts of surface area, the denatured state at high temperature and low urea concentration is more compact. To disentangle these two effects, we use destabilizing mutations that shift the Tm and Cm. We find that the compaction of the denatured state is related to changing temperature as the different variants of acyl-coenzyme A binding protein have similar m-values when they are at the same temperature but different urea concentration. These results have important implications for protein folding and stability under different environmental conditions.

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO−), were explored. G6PDH was incubated with AAPH (2,2′ -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO−. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 μM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO− induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO−. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO−. LC-MS analysis provided evidence for ONOO−-mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity. [Display omitted] • Oxidation and inactivation of G6PDH induced by ROO● and ONOO−, was investigated. • Both oxidants decreased enzymatic activity, as evidenced by NADPH formation. • ROO●-mediated G6PDH inactivation is related to protein unfolding and aggregation. • ONOO− induces extensive Met and Tyr oxidation without altering protein conformation. • Chloramine T studies indicate a key role for Met oxidation in G6PDH inactivation. [ABSTRACT FROM AUTHOR]

Protein‐polymer conjugates are a blooming class of hybrid systems with high biomedical potential. Despite a plethora of papers on their biomedical properties, the physical–chemical characterization of many protein‐polymer conjugates is missing. Here, we evaluated the thermal stability of a set of fully‐degradable polyphosphoester‐protein conjugates by variable temperature circular dichroism, a common but powerful technique. We extensively describe their thermodynamic stability in different environments (in physiological buffer or in presence of chemical denaturants, e.g., acid or urea), highlighting the protective role of the polymer in preserving the protein from denaturation. For the first time, we propose a simple but effective protocol to achieve useful information on these systems in vitro, useful to screen new samples in their early stages. [ABSTRACT FROM AUTHOR]

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c. [ABSTRACT FROM AUTHOR]