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Biomaterials synthesized from cross-linked folded proteins have untapped potential for biocompatible, resilient, and responsive implementations, but face challenges due to costly molecular refinement and limited understanding of their mechanical response. Under a stress vector, these materials combine the gel-like response of cross-linked networks with the mechanical unfolding and extension of proteins from well-defined 3D structures to unstructured polypeptides. Yet the nanoscale dynamics governing their viscoelastic response remains poorly understood. This lack of understanding is further exacerbated by the fact that the mechanical stability of protein domains depends not only on their structure, but also on the direction of the force vector. To this end, here we propose a coarse-grained network model based on the physical characteristics of polyproteins and combine it with the mechanical unfolding response of protein domains, obtained from single molecule measurements and steered molecular dynamics simulations, to explain the macroscopic response of protein-based materials to a stress vector. We find that domains are about 10-fold more stable when force is applied along their end-to-end coordinate than along the other tethering geometries that are possible inside the biomaterial. As such, the macroscopic response of protein-based materials is mainly driven by the unfolding of the node-domains and rearrangement of these nodes inside the material. The predictions from our models are then confirmed experimentally using force-clamp rheometry. This model is a critical step toward developing protein-based materials with predictable response and that can enable applications for shape memory and energy storage and dissipation.

AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP's ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX's ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains., (© 2024. The Author(s).)

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We explore how a protein's native structure determines its unfolding process. We examine how the local structural features, like shear, and the global structural properties, like the number of soft modes, change during unfolding. Simulations are performed using a Gaussian Network Model (GNM) with bond breaking for both thermal and force-induced unfolding scenarios. We find that unfolding starts in areas of high shear in the native structure and progressively spreads to the low shear regions. Interestingly, analysis of single domain protein families (Chymotrypsin inhibitor and Barnase) reveal that proteins with distinct unfolding pathways exhibit divergent behavior of the number of soft modes during unfolding. This suggests that the number of soft modes might be a valuable tool for understanding thermal unfolding pathways. Additionally, we found a strong link between a protein's overall structural similarity (TM-score) and its unfolding pathways, highlighting the importance of the native structure in determining how a protein unfolds., (© 2024. The Author(s).)

Escherichia coli ClpB and Saccharomyces cerevisiae Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed. We reported that Hsp104 and ClpB are non-processive on unfolded model substrates. Others have reported that ClpB is able to processively translocate a mechanically unfolded polypeptide chain at rates over 240 amino acids (aa) per second. Here, we report the development of a single turnover stopped-flow fluorescence strategy that reports on processive protein unfolding catalyzed by ClpB. We show that when translocation catalyzed by ClpB is challenged by stably folded protein structure, the motor enzymatically unfolds the substrate at a rate of ~0.9 aa s -1 with a kinetic step-size of ~60 amino acids at sub-saturating [ATP]. We reconcile the apparent controversy by defining enzyme catalyzed protein unfolding and translocation as two distinct reactions with different mechanisms of action. We propose a model where slow unfolding followed by fast translocation represents an important mechanistic feature that allows the motor to rapidly translocate up to the next folded region or rapidly dissociate if no additional fold is encountered., Competing Interests: JB, AL consultant for Nitrase Therapeutics, LI No competing interests declared, (© 2024, Banwait et al.)

Understanding the binding details of a small-molecule drug to a protein in its partially unfolded state is important for drug delivery as it provides insight into the overall drug-binding ability of the protein, even when the majority of binding pockets in its unfolded state are impaired. The interaction of partially unfolded proteins with drugs remains poorly understood due to a lack of structural information on proteins in their partially unfolded states. Here, we studied the interaction between serum albumin (bovine serum albumin (BSA) as a model system), an abundant protein in blood serum that is an effective carrier for numerous known drugs, and a nonsteroidal anti-inflammatory drug (NSAID) naproxen (NPS) using various spectroscopic and computational methods. Molecular dynamics simulations starting from the drug-unbound state and performed at physiological and higher temperatures revealed novel hydrophobic sites on the BSA surface. We analyzed the BSA-NPS interaction in the presence and absence of the cationic organized assembly CTAB and two oligosaccharides (β-CD and 2-HP-β-CD) at different excitation wavelengths. The solvation dynamics of BSA under NPS-bound conditions became ∼4.6% faster. Oligosaccharides were found to increase the solubility of NPS by providing a hydrophobic environment for the formation of inclusion complexes through host-guest interactions. These findings provide a comprehensive overview and uncover the binding model and mechanism of interaction of NPS with BSA, revealing hydrophobic and electrostatic interactions and hydrogen bonds required for BSA to bind NPS at these noncanonical sites. The molecular-level understanding of the binding mechanism of commonly used NSAIDs like NPS with partially unfolded BSA will be useful in designing pharmaceutically important molecules with efficient loading and delivery properties.

The relative magnitudes of noncovalent stabilization energies or the coupling free energies in folded proteins are anisotropically distributed, uniquely influencing folding and functional behaviors. In this regard, the fructose repressor (FruR) DBD belonging to the LacR repressor family harbors a three-residue insertion─KQY─between the canonical second and third helices. This sequence insertion promotes a strong Tyr-Tyr stacking interaction that is not observed in related homologues. Combining experiments with simulations, we show that the Tyr-Tyr stacking contributes to a decoupled unfolding due to the localization of a large part of the stabilization energy in this specific structural region. This leads to melting temperatures from different probes spanning nearly 10 K, while concomitantly stabilizing a partially structured intermediate state. Disruption of the aromatic stacking interaction via an alanine mutation promotes a molten-globular state whose native ensemble is replete with non-native interactions while displaying enhanced thermodynamic fluctuations and minimal calorimetric cooperativity. Surprisingly, the molten-globular variant of FruR DBD binds to the operator site on DNA with an affinity similar to that of the wild-type but with altered secondary-structure characteristics in the bound state, underscoring the chaperone-like role of DNA through its large negative electrostatic potential. FruR DBD thus appears to be at the verge of disorder as expected of an entropically destabilizing three-residue insertion but is rescued by the aromatic stacking interaction that distinctly dictates the finer details of stability, cooperativity, and binding.

Mechanical force controls the opening and closing of mechanosensitive ion channels atop the hair bundles of the inner ear. The filamentous tip link connecting transduction channels to the tallest neighboring stereocilium modulates the force transmitted to the channels and thus changes their probability of opening. Each tip link comprises four molecules: a dimer of protocadherin 15 (PCDH15) and a dimer of cadherin 23, all of which are stabilized by Ca 2+ binding. Using a high-speed optical trap to examine dimeric PCDH15, we find that the protein's mechanical properties are sensitive to Ca 2+ and that the molecule exhibits limited unfolding at a physiological Ca 2+ concentration. PCDH15 can therefore modulate its stiffness without undergoing large unfolding events under physiological conditions. The experimentally determined stiffness of PCDH15 accords with published values for the stiffness of the gating spring, the mechanical element that controls the opening of mechanotransduction channels. When PCDH15 exhibits a point mutation, V507D, associated with nonsyndromic hearing loss, unfolding events occur more frequently under tension and refolding events occur less often than for the wild-type protein. Our results suggest that the maintenance of appropriate tension in the gating spring is critical to the appropriate transmission of force to transduction channels, and hence to hearing., Competing Interests: Competing interests statement:Although co-author J.E.C. and reviewer M.S. are both affiliated with the University of Chicago, they have not collaborated within the last 4 y in any capacity.

Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation to subsequent sites. Here we present a cell-free nanoscale model that exploits compartmentalization strategies to carry out regulated protein unfolding and degradation. Our synthetic model comprises two connected DNA origami nanocompartments (each measuring 25 nm × 41 nm × 53 nm): one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97 within the first compartment, establishing a gateway mechanism that controls substrate recruitment, translocation and processing within the second compartment. Our data show that, whereas spatial confinement increases the rate of the individual reactions by up to tenfold, the physical connection of the compartmentalized enzymes into a chimera efficiently couples the two reactions and reduces off-target proteolysis by almost sixfold. Hence, our modular approach may serve as a blueprint for engineering artificial nanofactories with reshaped catalytic performance and functionalities beyond those observed in natural systems., (© 2024. The Author(s).)

Immunoglobulin G (IgG) comprises a significant portion of the protein corona that forms on biomaterial surfaces and holds a pivotal role in modulating host immune responses. To shed light on the important relationship between biomaterial surface functionality, IgG adsorption, and innate immune responses, we prepared, using plasma deposition, four surface coatings with specific chemistries, wettability, and charge. We found that nitrogen-containing coatings such as these deposited from allylamine (AM) and 2-methyl-2-oxazoline (POX) cause the greatest IgG unfolding, while hydrophilic acrylic acid (AC) surfaces allowed for the retention of the protein structure. Structural changes in IgG significantly modulated macrophage attachment, migration, polarization, and the expression of pro- and anti-inflammatory cytokines. Unfolded IgG on the POX and AM surfaces enhanced macrophage attachment, migration, extracellular trap release, and pro-inflammatory factors production such as IL-6 and TNF-α. Retention of IgG structure on the AC surface downregulated inflammatory responses. The findings of this study demonstrate that the retention of protein structure is an essential factor that must be taken into consideration when designing biomaterial surfaces. Our study indicates that using hydrophilic surface coatings could be a promising strategy for designing immune-modulatory biomaterials for clinical applications.

The combination of infrared spectroscopy (IR) and ion mobility mass spectrometry (IM-MS) has revealed that protein secondary structures are retained upon transformation from aqueous solution to the gas phase under gentle conditions. Yet the details about where and how these structural elements are embedded in the gas phase remain elusive. In this study, we employ long time scale molecular dynamics (MD) simulations to examine the extent to which proteins retain their solution structures and the impact of protonation state on the stability of secondary structures in the gas phase. Our investigation focuses on two well-studied proteins, myoglobin and β-lactoglobulin, representing typical helical and β-sheet proteins, respectively. Our simulations accurately reproduce the experimental collision cross section (CCS) data measured by IM-MS. Based on accurately reproducing previous experimental collision cross section data and dominant secondary structural species obtained from IM-MS and IR, we confirm that both proteins largely retain their native secondary structural components upon passing from aqueous solution to the gas phase. However, we observe significant reductions in secondary structure contents (19.2 ± 1.2% for myoglobin and 7.3 ± 0.6% for β-lactoglobulin) in specific regions predominantly composed of ionizable residues. Further mechanistic analysis suggests that alterations in protonation states of these residues after phase transition induce changes in their local interaction networks and backbone dihedral angles, which potentially promote the unfolding of secondary structures in the gas phase. We anticipate that similar protonation state induced unfolding may be observed in other proteins possessing distinct secondary structures. Further studies on a broader array of proteins will be essential to refine our understanding of protein structural behavior during the transition to the gas phase.

Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 5 2 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 6 1 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature ( T ∼20-95 °C). An increase in the protein charge states and collision cross sections (∼2750-8750 Å 2 and ∼3250-15,385 Å 2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrP C ) converts into misfolded abnormal scrapie prion (PrP Sc ) through post-translational modifications that increase β-sheet conformation. We recently demonstrated that HuPrP(90-231) thermal unfolding is partially irreversible and characterized by an intermediate state (β-PrPI), which has been revealed to be involved in the initial stages of PrP C fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90-231)) and full-length (HuPrP(23-231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90-231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90-231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to β-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.

Protein-surfactant interaction is a dynamic interplay of electrostatic and hydrophobic forces that ensues from the folding of a protein. We employ impedance spectroscopy (IS), a label-free method, to investigate the unfolding and refolding of human serum albumin (HSA), a globular plasma protein, in the presence of two surfactants: polysorbate-20 (Tween-20), a nonionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant. The equivalent electrical analog circuit was predicted from impedance spectra of HSA in an aqueous solution at physiological pH and room temperature, focusing on varying the concentration of codissolved surfactants. A change in the dielectric constant (ε ' ) and ionic conductivity (κ) is observed by comparing the surfactant-treated protein samples to the bare surfactant solutions to assess the conformational changes induced by surfactants in HSA. Far-UV circular dichroism analysis revealed a decrease in α-helices and an increase in β-sheets and random coils upon SDS addition, which were reversed by Tween-20. Dynamic light scattering supported the findings by measuring changes in the hydrodynamic diameter ( d h ) of HSA. Unfolding and refolding of HSA with surfactants were also observed through photoluminescence spectroscopy by examining the microenvironment surrounding the single tryptophan ( W ) within the protein, and the thermodynamic parameters were obtained using the modified Stern-Volmer equation. Our research explores the intriguing domain of protein-surfactant interactions, offering insights with promising applications across diverse biological processes and IS as a suitable alternative technique for investigating protein conformational changes by studying the electrical response of the samples.

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-NO 2 , synthetic precursors of R-ASC-MI, in cellular hypoxia. R-ASC-NO 2 revealed upregulated activities of nitroreductase, as well as increased hydrophobicity in live cells, under either chemical (NaN 3 ) induced or atmospheric (1 % O 2 ) 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., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Here we present TPPU_DSF (https://maciasnmr.net/tppu_dsf/). This is a free and open-source web application that opens, converts, fits, and calculates the thermodynamic parameters of protein unfolding from standard differential scanning fluorimetry (DSF) data in an automated manner. The software has several applications. In the context of screening compound libraries for protein binders, obtaining thermodynamic parameters provides a more robust approach to detecting hits than the changes in the melting temperature (T m ) alone, thereby helping to increase the number of positive hits in screening campaigns. Moreover, changes in ΔG u o indicate protein response to binding at lower compound concentrations than those in the T m , thereby reducing the costs associated with the amounts of protein and compounds required for the assays. Also, by adding thermodynamic information to the T m comparison, the software can contribute to the optimization of protein constructs and buffer conditions, a common practice before structural and functional projects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Most proteins fold during biosynthesis on the ribosome 1 , and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies 2-10 . The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19 F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol -1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding., (© 2024. The Author(s).)

Liquid egg products are typically exposed to a defined thermal load to achieve the required safety level, under which their functional properties can be adversely affected. In this study, manothermosonication (MTS) (132 μm, 300 kPa) was investigated as alternative preservation for liquid whole egg (LWE) compared to thermal pasteurisation (60 °C, 3.5 min), assessing results against untreated (fresh) LWE in terms of selected physico-chemical properties. Results showed that MTS resulted in improved LWE foaming properties, increasing foam capacity by a 3.2-fold factor compared to thermal treatment. Emulsion stability was also enhanced after MTS, exhibiting smaller droplet size, and a higher elasticity of gels was obtained. Regarding the protein properties, favourable protein changes (protein unfolding) were identified for MTS through direct (asymmetric flow field flow fractionation) and indirect (surface hydrophobicity and sulfhydryl group content) measurements. In addition, an increase in protein solubility of 11.4 % was observed in MTS compared to thermal treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this study, the interaction of the human hemoglobin with cost effective and chemically fabricated CdS quantum dots (QDs) (average sizes ≈3nm) has been investigated. The semiconductor QDs showed maximum visible absorption at 445 nm with excitonic formation and band gap of ≈ 2.88 eV along with hexagonal crystalline phase. The binding of QDs-Hb occurs through corona formation to the ground sate complex formation. The life time of the heme pocket binding and reorganization were found to be t 1  = 43 min and t 2  = 642 min, respectively. The emission quenching of the Hb has been indicated large energy transfer between CdS QDs and Hb with tertiary deformation of Hb. The binding thermodynamics showed highly exothermic nature. The ultrafast decay during corona formation was studied from TCSPC. The results showed that the energy transfer efficiency increases with the increase of the QDs concentration and maximum ≈71.5 % energy transfer occurs and average ultrafast lifetime varies from 5.45 ns to1.51 ns. The deformation and unfolding of the secondary structure of Hb with changes of the α-helix (≈74 % to ≈51.07 %) and β-sheets (≈8.63 % to ≈10.25 %) have been observed from circular dichroism spectrum. The SAXS spectrum showed that the radius of gyration of CdS QDs-Hb bioconjugate increased (up to 23 ± 0.45 nm) with the increase of the concentration of QDs compare with pure Hb (11 ± 0.23 nm) and Hb becoming more unfolded., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein folding and unfolding experiments are interpreted under the assumption of microscopic reversibility, that is, that at equilibrium one process is the reverse of the other. Single-domain proteins illustrate the validity of such an interpretation, although reversibility does not necessarily hold under the different conditions typically used for folding and unfolding experiments. In fact, more complex proteins, which often exhibit irreversible unfolding, are generally considered not amenable to folding kinetics studies. Here, the X11 PDZ1-PDZ2 tandem repeat allows us to reveal the different folding and unfolding pathways at play under different experimental conditions, thus reconciling the apparent contradiction between theory and experiment., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding., (© 2024. The Author(s).)

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates., Competing Interests: Competing interests statement:The authors declare no competing interest.

Kinetic stability is thought to be an attribute of proteins that require a long lifetime, such as the transporter of thyroxine and holo retinol-binding protein or transthyretin (TTR) functioning in the bloodstream, cerebrospinal fluid, and vitreous humor. TTR evolved from ancestral enzymes known as TTR-related proteins (TRPs). Here, we develop a rate-expansion approach that allows unfolding rates to be measured directly at low denaturant concentration, revealing that kinetic stability exists in the Escherichia coli TRP (EcTRP), even though the enzyme structure is more energetically frustrated and has a more mutation-sensitive folding mechanism than human TTR. Thus, the ancient tetrameric enzyme may already have been poised to mutate into a kinetically stable human transporter. An extensive mutational study that exchanges residues at key sites within the TTR and EcTRP dimer-dimer interface shows that tyrosine 111, replaced by a threonine in TTR, is the gatekeeper of frustration in EcTRP because it is critical for function. Frustration, virtually absent in TTR, occurs at multiple sites in EcTRP and even cooperatively for certain pairs of mutations. We present evidence that evolution at the C terminus of TTR was a compensatory event to maintain the preexisting kinetic stability while reducing frustration and sensitivity to mutation. We propose an "overcompensation" pathway from EcTRPs to functional hybrids to modern TTRs that is consistent with the biophysics discussed here. An alternative plausible pathway is also presented., Competing Interests: Competing interests statement:The authors declare no competing interest.

Even 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.

Vinculin binds to specific sites of mechanically unfolded talin rod domains to reinforce the coupling of the cell's exterior to its force generation machinery. Force-dependent vinculin-talin complexation and dissociation was previously observed as contraction or extension of the unfolded talin domains respectively using magnetic tweezers. However, the structural mechanism underlying vinculin recognition of unfolded vinculin binding sites (VBSs) in talin remains unknown. Using molecular dynamics simulations, we demonstrate that a VBS dynamically refolds under force, and that vinculin can recognize and bind to partially unfolded VBS states. Vinculin binding enables refolding of the mechanically strained VBS and stabilizes its folded α-helical conformation, providing resistance against mechanical stress. Together, these results provide an understanding of a recognition mechanism of proteins unfolded by force and insight into the initial moments of how vinculin binds unfolded talin rod domains during the assembly of this mechanosensing meshwork., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mykuliak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ion mobility-mass spectrometry (IM-MS) has become a technology deployed across a wide range of structural biology applications despite the challenges in characterizing closely related protein structures. Collision-induced unfolding (CIU) has emerged as a valuable technique for distinguishing closely related, iso-cross-sectional protein and protein complex ions through their distinct unfolding pathways in the gas phase. With the speed and sensitivity of CIU analyses, there has been a rapid growth of CIU-based assays, especially regarding biomolecular targets that remain challenging to assess and characterize with other structural biology tools. With information-rich CIU data, many software tools have been developed to automate laborious data analysis. However, with the recent development of new IM-MS technologies, such as cyclic IM-MS, CIU continues to evolve, necessitating improved data analysis tools to keep pace with new technologies and facilitating the automation of various data processing tasks. Here, we present CIUSuite 3, a software package that contains updated algorithms that support various IM-MS platforms and supports the automation of various data analysis tasks such as peak detection, multidimensional classification, and collision cross section (CCS) calibration. CIUSuite 3 uses local maxima searches along with peak width and prominence filters to detect peaks to automate CIU data extraction. To support both the primary CIU (CIU 1 ) and secondary CIU (CIU 2 ) experiments enabled by cyclic IM-MS, two-dimensional data preprocessing is deployed, which allows multidimensional classification. Our data suggest that additional dimensions in classification improve the overall accuracy of class assignments. CIUSuite 3 also supports CCS calibration for both traveling wave and drift tube IM-MS, and we demonstrate the accuracy of a new single-field CCS calibration method designed for drift tube IM-MS leveraging calibrant CIU data. Overall, CIUSuite 3 is positioned to support current and next-generation IM-MS and CIU assay development deployed in an automated format.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The blood protein Von Willebrand factor (VWF) is critical in facilitating arterial thrombosis. At pathologically high shear rates, the protein unfolds and binds to the arterial wall, enabling the rapid deposition of platelets from the blood. We present a novel continuum model for VWF dynamics in flow based on a modified viscoelastic fluid model that incorporates a single constitutive relation to describe the propensity of VWF to unfold as a function of the scalar shear rate. Using experimental data of VWF unfolding in pure shear flow, we fix the parameters for VWF's unfolding propensity and the maximum VWF length, so that the protein is half unfolded at a shear rate of approximately 5000 s - 1 . We then use the theoretical model to predict VWF's behaviour in two complex flows where experimental data are challenging to obtain: pure elongational flow and stenotic arterial flow. In pure elongational flow, our model predicts that VWF is 50% unfolded at approximately 2000 s - 1 , matching the established hypothesis that VWF unfolds at lower shear rates in elongational flow than in shear flow. We demonstrate the sensitivity of this elongational flow prediction to the value of maximum VWF length used in the model, which varies significantly across experimental studies, predicting that VWF can unfold between 2000 and 3200 s - 1 depending on the selected value. Finally, we examine VWF dynamics in a range of idealised arterial stenoses, predicting the relative extension of VWF in elongational flow structures in the centre of the artery compared to high shear regions near the arterial walls., (© 2024. The Author(s).)

Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological temperatures that is rapidly degraded in the cell. This mutation is present in about 90% of cystic fibrosis patients, hence there is great interest in compounds reversing its instability. We have previously reported the expression of the mutated protein at low temperature and its purification in detergent. Here we describe the use of the protein to screen compounds present in a library of Federal Drug Administration (FDA) - approved drugs and also in a small natural product library. The kinetics of unfolding of F508del CFTR at 37 °C were probed by the increase in solvent-exposed cysteine residues accessible to a fluorescent reporter molecule. This occurred in a bi-exponential manner with a major (≈60%) component of half-life around 5 min and a minor component of around 60 min. The faster kinetics match those observed for loss of channel activity of F508del CFTR in cells at 37 °C. Most compounds tested had no effect on the fluorescence increase, but some were identified that significantly slowed the kinetics. The general properties of these compounds, and any likely mechanisms for inducing stability in purified CFTR are discussed. These experimental data may be useful for artificial intelligence - aided design of CFTR-specific drugs and in the identification of stabilizing additives for membrane proteins (in general)., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAbs) are an essential class of therapeutic proteins for the treatment of cancer, autoimmune and rare diseases. During their production, storage, and administration processes, these proteins encounter various stressors such as temperature fluctuations, vibrations, and light exposure, able to induce chemico-physical modifications to their structure. Viral inactivation is a key step in downstream processes, and it is achieved by titration of the mAb at low pH, followed by neutralization. The changes of the pH pose a significant risk of unfolding and subsequent aggregation to proteins, thereby affecting their manufacturing. This study aims to investigate whether a combined exposure to light during the viral inactivation process can further affect the structural integrity of Ipilimumab, a mAb primarily used in the treatment of metastatic melanoma. The biophysical and biochemical characterization of Ipilimumab revealed that pH variation is a considerable risk for its stability with irreversible unfolding at pH 2. The threshold for Ipilimumab denaturation lies between pH 2 and 3 and is correlated with the loss of the protein structural cooperativity, which is the most critical factor determining the protein refolding. Light has demonstrated to exacerbate some local and global effects making pH-induced exposed regions more vulnerable to structural and chemical changes. Therefore, specific precautions to real-life exposure to ambient light during the sterilization process of mAbs should be considered to avoid loss of the therapeutic activity and to increase the yield of production. Our findings underscore the critical role of pH optimization in preserving the structural integrity and therapeutic efficacy of mAbs. Moreover, a detailed conformational study on the structural modifications of Ipilimumab may improve the chemico-physical knowledge of this effective drug and suggest new production strategies for more stable products under some kind of stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for Intrinsic Tryptophan Fluorescence Emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (< 5 mg/mL, equivalent to 0.8 mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (> 40 mg/mL, equivalent to 6.1 mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223 mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1 %) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nonenzymatic glycation (NEG) unfolds and crosslinks proteins, resulting in aggregation. Label-free evaluation of such structural changes, without disturbing molecular integrity, would be beneficial for understanding the fundamental mechanisms of protein aggregation. The current study demonstrates the assessment of NEG-induced protein aggregation by combining autofluorescence (AF) spectroscopy and imaging. The methylglyoxal (MG) induced protein unfolding and the formation of cross-linking advanced glycation end-products (AGEs) leading to aggregation were evaluated using deep-UV-induced-autofluorescence (dUV-AF) spectroscopy in proteins with distinct structural characteristics. Since the AGEs formed on proteins are fluorescent, the study demonstrated the possibility of autofluorescence imaging of NEG-induced protein aggregates. Autofluorescence spectroscopy can potentially reveal molecular alterations such as protein unfolding and cross-linking. In contrast, AGE-based autofluorescence imaging offers a means to visually explore the structural arrangement of aggregates, regardless of whether they are amyloid or non-amyloid in nature.

The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection., (© 2024. The Author(s).)

Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H] 12+ ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an E lab energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H] 12+ EF-Tu ion showed similar qualitative behavior to the experimental results.

Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of interest using OT, the protein must be flanked with two double stranded DNA (dsDNA) handles. However, coupling dsDNA handles to the protein is often of low yield, representing a bottleneck in OT experiments. Here, we report a handle-free, all-protein-based OT method for investigating protein folding/unfolding dynamics. In this new method, we employed disordered elastin-like polypeptides (ELPs) as a molecular linker and the mechanically stable cohesin-dockerin (Coh-Doc) pair as the prey-bait system to enable the efficient capture and stretching of individual protein molecules. This novel approach was validated by using model proteins NuG2 and RTX-v, yielding experimental results comparable to those obtained by using the dsDNA handle approach. This new method provides a streamlined and efficient OT approach to investigate the folding-unfolding dynamics of proteins at the single molecule level, thus expanding the toolbox of OT-based single molecule force spectroscopy.

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short c U 50 T is introduced. Analyzing a set of ene reductases, c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature., (© 2024. The Author(s).)

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15 N and 13 C HSQC NMR spectroscopy to gain structural insight into the CuSO 4 -mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

The mechanical stability of proteins is crucial for biological processes. To understand the mechanical functions of proteins, it is important to know the protein structure and mechanical properties. Protein mechanics is usually investigated through force spectroscopy experiments and simulations that probe the forces required to unfold the protein of interest. While there is a wealth of data in the literature on force spectroscopy experiments and steered molecular dynamics simulations of forced protein unfolding, this information is spread and difficult to access by non-experts. Here, we introduce MechanoProDB, a novel web-based database resource for collecting and mining data obtained from experimental and computational works. MechanoProDB provides a curated repository for a wide range of proteins, including muscle proteins, adhesion molecules and membrane proteins. The database incorporates relevant parameters that provide insights into the mechanical stability of proteins and their conformational stability such as the unfolding forces, energy landscape parameters and contour lengths of unfolding steps. Additionally, it provides intuitive annotations of the unfolding pathways of each protein, allowing users to explore the individual steps during mechanical unfolding. The user-friendly interface of MechanoProDB allows researchers to efficiently navigate, search and download data pertaining to specific protein folds or experimental conditions. Users can visualize protein structures using interactive tools integrated within the database, such as Mol*, and plot available data through integrated plotting tools. To ensure data quality and reliability, we have carefully manually verified and curated the data currently available on MechanoProDB. Furthermore, the database also features an interface that enables users to contribute new data and annotations, promoting community-driven comprehensiveness. The freely available MechanoProDB aims to streamline and accelerate research in the field of mechanobiology and biophysics by offering a unique platform for data sharing and analysis. MechanoProDB is freely available at https://mechanoprodb.ibdm.univ-amu.fr., (© The Author(s) 2024. Published by Oxford University Press.)

A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M 2/3 ) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, β-lactoglobulin (2 disulfide bridges), calmodulin (Ca 2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TW CCS N2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TW CCS N2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH 1.5 shift, pH 12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH  12 shift treatment. In addition, pH 1.5 shift modified BSFLA tended to form β-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH 12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH 12 -shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 The Protein Society.)

Mechanical properties of proteins that have a crucial effect on their operation. This study used a molecular dynamics simulation package to investigate rubredoxin unfolding on the atomic scale. Different simulation techniques were applied, and due to the dissociation of covalent/hydrogen bonds, this protein demonstrates several intermediate states in force-extension behavior. A conceptual model based on the cohesive finite element method was developed to consider the intermediate damages that occur during unfolding. This model is based on force-displacement curves derived from molecular dynamics results. The proposed conceptual model is designed to accurately identify bond rupture points and determine the associated forces. This is achieved by conducting a thorough comparison between molecular dynamics and cohesive finite element results. The utilization of a viscoelastic cohesive zone model allows for the consideration of loading rate effects. This rate-dependent model can be further developed and integrated into the multiscale modeling of large assemblies of metalloproteins, providing a comprehensive understanding of mechanical behavior while maintaining a reduced computational cost., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A substantial portion of various organisms' proteomes comprises intrinsically disordered proteins (IDPs) that lack a defined three-dimensional structure. These IDPs exhibit a diverse array of conformations, displaying remarkable spatiotemporal heterogeneity and exceptional conformational flexibility. Characterizing the structure or structural ensemble of IDPs presents significant conceptual and methodological challenges owing to the absence of a well-defined native structure. While databases such as the Protein Ensemble Database (PED) provide IDP ensembles obtained through a combination of experimental data and molecular modeling, the absence of reaction coordinates poses challenges in comprehensively understanding pertinent aspects of the system. In this study, we leverage the energy landscape visualization method (JCTC, 6482, 2019) to scrutinize four IDP ensembles sourced from PED. ELViM, a methodology that circumvents the need for a priori reaction coordinates, aids in analyzing the ensembles. The specific IDP ensembles investigated are as follows: two fragments of nucleoporin (NUL: 884-993 and NUS: 1313-1390), yeast sic 1 N-terminal (1-90), and the N-terminal SH3 domain of Drk (1-59). Utilizing ELViM enables the comprehensive validation of ensembles, facilitating the detection of potential inconsistencies in the sampling process. Additionally, it allows for identifying and characterizing the most prevalent conformations within an ensemble. Moreover, ELViM facilitates the comparative analysis of ensembles obtained under diverse conditions, thereby providing a powerful tool for investigating the functional mechanisms of IDPs.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Acylphosphatase (AcP) is an enzyme which catalyses the hydrolysis of acylphosphate. The binding with the phosphate ion (Pi) assumes significance in preserving both the stability and enzymatic activity of AcP. While previous studies using single molecule force spectroscopy explored the mechanical properties of AcP, the influence of Pi on its folding and unfolding dynamic behaviors remains unexplored. In this work, using stable magnetic tweezers, we measured and compared the force-dependent folding and unfolding rates of AcP in the Tris buffer and phosphate buffer within a force range from 2 pN to 40 pN. We found that Pi exerts no discernible effect on the folding dynamics but consistently decreases the force-dependent unfolding rate of AcP by a constant ratio across the entire force spectrum. The free energy landscapes of AcP in the absence and presence of Pi are constructed. Our results reveal that Pi selectively binds to the native state of AcP, stabilizing it and suggesting the general properties of specific ligand-receptor interactions.