Unfolding Behavior Research Articles

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations.

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling.

Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations.

A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

Atomic-Scale View of Protein-PEG Interactions that Redirect the Thermal Unfolding Pathway of PEGylated Human Galectin-3.

PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Atomic‐Scale View of Protein‐PEG Interactions that Redirect the Thermal Unfolding Pathway of PEGylated Human Galectin‐3

AbstractPEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein‐PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin‐3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein‐PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

Free-energy landscape of two-state protein acylphosphatase with large contact order revealed by force-dependent folding and unfolding dynamics.

Acylphosphatase (AcP) is a small protein with 98 amino acid residues that catalyzes the hydrolysis of carboxyl-phosphate bonds. AcP is a typical two-state protein with slow folding rate due to its relatively large contact order in the native structure. The mechanical properties and unfolding behavior of AcP has been studied by atomic force microscope. Here using stable magnetic tweezers, we measured the force-dependent folding rates within a force range 1-3 pN, and unfolding rates 15-40 pN. The obtained unfolding rates show different force sensitivities at forces below and above ∼27 pN, which determines a free-energy landscape with two energy barriers. Our results indicate that the free-energy landscape of small globule proteins have general Bactrian camel shape, and large contact order of the native state produces a high barrier dominate at low forces.

Deployment and surface accuracy of regularly creased membranes

Creases are highly localized regions ubiquitous across different length scales in low-dimensional natural and engineering systems. Their presence strongly influences the mechanical response and surface accuracy of creased membrane materials and structures. In this paper, we study the deployment of folded sheets composed of an arbitrary number of non-interacting and parallel creases. We develop a mathematical formulation that describes the nonlinear mechanics of systematically creased membranes composed of a single or multiple folds, and predicts their surface accuracy during unfolding. The proposed solution shows the contribution of membrane bending and crease energies during deployment, and reveals the presence of two dimensionless parameters that govern the unfolding behaviour. Sensitivity analyses are also performed to assess the influence of the crease geometry and constitutive behaviour. The analytical predictions are validated through finite element analyses and deployment tests performed on thin films with one, two and three fold lines, where imaging techniques are employed to quantify deformation. The excellent agreement between theoretical and experimental results testifies that the developed formulation represents a precise tool to assess the tensioning of creased membranes, with applications ranging from origami metamaterials to lightweight space structures where precise shape control is paramount.

FORMULATION AND INVITRO EVALUATION OF PRAZOSIN HYDROCHLORIDE UNFOLDING TYPE GASTRO RETENTIVE FILM

The present work is based on the formulation and In-vitro evaluation of a gastroretentive mucoadhesvie based drug delivery system containing prazosin hydrochloride for controlled release. It consists of a drug loaded polymeric film folded into a hard gelatin capsule. After administration film unfolds and its swelling and bioadhesion to the gastric mucosa. Prazosin hydrochloride, a histamine H2 receptor antagonist used for gastroesophageal reflux disease (GERD), duodenal ulcer and gastric ulcer. Prazosin hydrochloride absorbed only in the initial part of gastro intestinal tract (GIT) and has less bioavailability. Thus by retaining the drug in the gastric region improves its bioavailability. Films were prepared by solventcasting method using HPMC K4M, and Carbopol 971P NF as polymers and PEG 400 as the plasticizer. The prepared film were evaluated for various parameters such as film thickness, folding endurance, uniformity of weight, surface pH, determination of drug content, moisture content, swelling index, In-vitro mucoadhesive study retention time, In-vitro unfolding behavior and In-vitro drug release studies and drug release kinetics. Differential scanning calorimetry revealed there were no polymorphic changes in drug as well as polymers during the formulation of polymeric film. Optimized formulation showed 99.02 % drug release at the end of 12 hrs and it follows the Korsmeyer-peppas kinetics model of drug release. Keywords: Gastroretentive mucoadhesive film, Solvent casting method, Prazosin hydrochloride

Formulation and Pharmacokinetic Evaluation of Ethyl Cellulose/HPMC-Based Oral Expandable Sustained Release Dosage of Losartan Potassium.

Prolonged retention of losartan potassium in the upper gastrointestinal tract is anticipated to increase its absorption and exposure to CYP450 enzyme subfamilies, undertaking its conversion to more potent (10-40 times) active metabolite, losartan carboxylic acid (LCA). Consistent with this, hydroxypropyl methylcellulose K4M/ethyl cellulose-based novel expandable films (EFs) containing losartan potassium (LP) suitable for prolonged retention in the stomach were developed. The films were prepared by solvent casting method. USP type II dissolution apparatus (0.1N HCl, 37°C, 100rpm) was used to perform the dissolution testing (drug release, unfolding behavior, film integrity, erosion, and water uptake) of the films. In vivo pharmacokinetic studies were carried out in rabbits. An HPLC-UV method was used for the quantification of the drug and its active metabolite in plasma. These folded films placed inside hard gelatin capsule shells unfolded to full dimensions in dissolution medium and provided sustained drug release throughout 12h. The plasma drug concentration-time curves obtained from the in vivo studies were used to determine pharmacokinetic parameters, such as area under the plasma drug concentration-time curve(AUC), area under first moment curve(AUMC), mean residence time (MRT), Cmax, Tmax, t1/2, ke, and Fr in comparison with that of the market formulation, Cozaar®. The novel EFs significantly changed the pharmacokinetic parameters of the drug and its active metabolite. The apparent elimination rate constant (ke) significantly decreased, while MRT and elimination half-life (t1/2) increased in both cases. The relative bioavailabilities (Fr) of both LP and E3174 using the novel formulation were higher than that of Cozaar®.

High-Pressure, Low-Temperature Induced Unfolding and Aggregation of Monoclonal Antibodies: Role of the Fc and Fab Fragments.

The effects of high pressure and low temperature on the stability of two different monoclonal antibodies (MAbs) were examined in this work. Fluorescence and small-angle neutron scattering were used to monitor the in situ effects of pressure to infer shifts in tertiary structure and characterize aggregation prone intermediates. Partial unfolding was observed for both MAbs, to different extents, under a range of pressure/temperature conditions. Fourier transform infrared spectroscopy was also used to monitor ex situ changes in secondary structure. Preservation of native secondary structure after incubation at elevated pressures and subzero ° C temperatures was independent of the extent of tertiary unfolding and reversibility. Several combinations of pressure and temperature were also used to discern the respective contributions of the isolated Ab fragments (Fab and Fc) to unfolding and aggregation. The fragments for each antibody showed significantly different partial unfolding profiles and reversibility. There was not a simple correlation between stability of the full MAb and either the Fc or Fab fragment stabilities across all cases, demonstrating a complex relationship to full MAb unfolding and aggregation behavior. That notwithstanding, the combined use of spectroscopic and scattering techniques provides insights into MAb conformational stability and hysteresis in high-pressure, low-temperature environments.

Unfolding behavior of self-folded boron nitride nanosheets inducing ductility of cementitious composites

A classical molecular dynamics simulation is adopted to study unfolding behavior of self-folded boron nitride nanosheets (BNNSs) in the cementitious composite. The simulation results show that unfoldings of the self-folded BNNSs initiate at the interface between the interlayers of the self-folded BNNSs because this interface is weaker than the BNNS-tobermorite interface under individual tensile and shear deformation. The interfacial microstructure shows that the ends of the self-folded BNNSs are embedded into the rugged surfaces of tobermorite, which exhibits a mechanical interlocking effect on the BNNSs-tobermorite interface. Furthermore, the existence of the hydrogen bonds at the BNNS-tobermorite interface yield a robust interfacial interaction between BNNSs and tobermorite. The critical peeling forces required to peel the adhesion between BNNS and tobermorite and between interlayers of self-folded BNNSs are obtained from a continuum model, which gives insights into the unfolding initiation of the self-folded BNNSs. The progressive peeling at the BNNS-tobermorite interface allows the self-folded BNNSs to maintain mechanical reinforcement in tobermorite under a large deformation at the atomistic scale. The atomistic scale reinforcement of the self-folded BNNSs in the deformation capacity and mechanical properties of cementitious composites significantly improves the ductility of cementitious composites.

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.

Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes.

Signaling proteins are composed of conserved protein interaction domains that serve as allosteric regulatory elements of enzymatic or binding activities. The ubiquitous, structurally conserved cyclic nucleotide binding (CNB) domain is found covalently linked to proteins with diverse folds that perform multiple biological functions. Given that the structures of cAMP-bound CNB domains in different proteins are very similar, it remains a challenge to determine how this domain allosterically regulates such diverse protein functions and folds. Instead of a structural perspective, we focus our attention on the energy landscapes underlying the CNB domains and their responses to cAMP binding. We show that optical tweezers is an ideal tool to investigate how cAMP binding coupled to interdomain interactions remodel the energy landscape of the regulatory subunit of protein kinase A (PKA), which harbors two CNB domains. We mechanically manipulate and probe the unfolding and refolding behavior of the CNB domains as isolated structures or selectively as part of the PKA regulatory subunit, and in the presence and absence of cAMP. Optical tweezers allows us to dissect the changes in the energy landscape associated with cAMP binding, and to examine the allosteric interdomain interactions triggered by the cyclic nucleotide. This single molecule approach can be used to study other modular, multidomain signaling proteins found in nature.

New insights into the folding-unfolding mechanism and conformations of cytochrome C.

Metalloproteins account for over one-third of all proteins in nature and play important roles in biological processes. The formation of the native structures of metalloproteins requires not only the correct folding of the polypeptide chains but also the proper incorporation of metal cofactors. Understanding the folding mechanism of metalloproteins has been challenging. Horse heart cytochrome C (cytc) is a classical model system for protein folding studies. Although a large number of ensemble studies have been carried out to characterize the folding mechanism of cytc, there is still a significant debate on the folding mechanism and the existence of the proposed “foldons”. Here, we used single-molecule optical tweezers to probe the mechanical folding–unfolding behaviors of cytc at the single-molecule level. By directly monitoring the folding and unfolding of holo-cytc, we revealed novel insights into the folding of cytc. Our results showed that the structural elements that are distant from the N- and C-termini can exist as a short-lived intermediate, a finding that contrasts with the general belief that the folding and packing of the N- and C-terminal helices are prerequisites for the folding of other structural elements in cytc. In addition, our results present strong evidence that apo-cytc, which has been long believed to be a random coil, is not a true random coil, and weak interactions within the unfolded polypeptide chain exist. Our results bring new insights into our understanding of the folding mechanisms of heme proteins as well as the role of heme in the folding process.

Ultra-light kirigami lantern chain for superior impact mitigation

Kirigami-based metamaterials are demonstrated to possess peculiar mechanical behaviors via carefully engineered architectures. This paper reports on the comprehensive investigation of a novel kirigami-based lantern chain. We tailor and fabricate the kirigami lanterns using simple paper sheets yet achieve an exceptional impact mitigation capability (orders of magnitude lower transmission compared to various available metamaterials). Detailed experimental and numerical exploration uncovers that the unique folding–unfolding behavior of kirigami lanterns during stress wave propagation yields energy redistribution and storage in different cells. Accordingly, an expanded waveform with decreasing amplitude and oscillating tails is achieved within the 1D chain, largely mitigating the impact. Meanwhile, the plastic deformation of paper materials also contributes to outstanding performances. Based on a validated finite element (FE) model, the governing laws of critical parameters, including impact energy, cell number, petal number, and hinge number, are systematically explored, where we find that the mitigation effect is also satisfactory even at the increased impact energy scenarios. Furthermore, an adaptable design of kirigami chain length and other geometric parameters is exploited to realize highly efficient and controllable mitigation when subjected to specific impact energy. This paper illustrates a new route to designing superior impact mitigation structures with ultra-lightweight materials, offering insights for innovation of next-generation impact protection strategies in the automotive and aerospace industries.

A Dual‐Origami Design that Enables the Quasisequential Deployment and Bending Motion of Soft Robots and Grippers

Soft fluidic actuators produce continuous and life‐like motions that are intrinsically safe, but current designs are not yet mature enough to enable large deployment with high force and low‐cost fabrication methods. Herein, soft fluidic actuators with two superimposed origami architectures are reported. Driven by a fluid input, the presented dual‐origami soft actuators produce quasisequential deployment and bending motion that is guided by unsymmetric unfolding of low‐stretchable origami components. The dominance between the deployment and bending can be shifted by varying the unfolding behavior, enabling preprogramming of the motion. The proposed origami‐inspired soft actuators are directly fabricated by low‐cost fused deposition modeling 3D printing and subjected to heat treatment postprocessing to enhance the fluid sealing performance. Finally, soft gripper applications are presented and they successfully demonstrate gripping tasks where each requires strength, delicacy, precision, and dexterity. The dual‐origami approach offers a design guidance for soft robots to embody grow‐and‐retract motion with a small initial form factor, promising for applications in next‐generation soft robotic systems. An interactive preprint version of the article can be found here: https://www.authorea.com/doi/full/10.22541/au.163698906.68661340.

The role of water in the reversibility of thermal denaturation of lysozyme in solid and liquid states

Although unfolding of protein in the liquid state is relatively well studied, its mechanisms in the solid state, are much less understood. We evaluated the reversibility of thermal unfolding of lysozyme with respect to the water content using a combination of thermodynamic and structural techniques such as differential scanning calorimetry, synchrotron small and wide-angle X-ray scattering (SWAXS) and Raman spectroscopy. Analysis of the endothermic thermal transition obtained by DSC scans showed three distinct unfolding behaviors at different water contents. Using SWAXS and Raman spectroscopy, we investigated reversibility of the unfolding for each hydration regime for various structural levels including overall molecular shape, secondary structure, hydrophobic and hydrogen bonding interactions. In the substantially dehydrated state below 37 wt% of water the unfolding is an irreversible process and can be described by a kinetic approach; above 60 wt% the process is reversible, and the thermodynamic equilibrium approach is applied. In the intermediate range of water contents between 37 wt% and 60 wt%, the system is phase separated and the thermal denaturation involves two processes: melting of protein crystals and unfolding of protein molecules. A phase diagram of thermal unfolding/denaturation in lysozyme - water system was constructed based on the experimental data.

Flow-regulated nucleation protrusion theory for collapsed polymers.

The globular-stretch transition of a collapsed polymer in low strain rate elongational flow is studied using polymeric protrusion kinetics scaling laws and numerical simulation. Results demonstrate the influence of fluid flow on the occurrence probability of long-length thermally nucleated polymeric protrusions, which regulate collapsed polymer unfolding in low strain rate flows. Further, we estimate that the globular-stretch transition rate (k_{s}) in low strain rate (∈[over ̇]) elongational flows varies as k_{s}∼e^{-α∈[over ̇]^{-1}}. Results here reveal that the existing approach of neglecting the effects of fluid flow on thermally nucleated protrusions distribution is not valid for analyzing polymer unfolding behavior in low strain rate flows. Neglecting such an effect overestimates the constant α in the scaling law of transition rate (k_{s}∼e^{-α∈[over ̇]^{-1}}) by a factor of 2.

Cubic and Hexagonal Mesophases for Protein Encapsulation: Structural Effects of Insulin Confinement

Monoolein-based cubic and hexagonal mesophases were investigated as matrices for insulin loading, at low pH, as a function of temperature and in the presence of increasing amounts of oleic acid, as a structural stabilizer for the hexagonal phase. Synchrotron small angle X-ray diffraction, rheological measurements, and attenuated total reflection-Fourier transform infrared spectroscopy were used to study the effects of insulin loading on the lipid mesophases and of the effect of protein confinement in the 2D- and 3D-lipid matrix water channels on its stability and unfolding behavior. We found that insulin encapsulation has only little effects both on the mesophase structures and on the viscoelastic properties of lipid systems, whereas protein confinement affects the response of the secondary structure of insulin to thermal changes in a different manner according to the specific mesophase: in the cubic structure, the unfolding toward an unordered structure is favored, while the prevalence of parallel β-sheets, and nuclei for fibril formation, is observed in hexagonal structures.

A coarse-grained mechanical model for folding and unfolding of tropoelastin with possible mutations

We propose a simple general framework to predict folding, native states, energy barriers, protein unfolding, as well as mutation induced diseases and other protein structural analyses. The model should not be considered as an alternative to classical approaches (Molecular Dynamics or Monte Carlo) because it neglects low scale details and rather focuses on global features of proteins and structural information. We aim at the description of phenomena that are out of the range of classical molecular modeling approaches due to the large computational cost: multimolecular interactions, cyclic behavior under variable external interactions, and similar. To demonstrate the effectiveness of the approach in a real case, we focus on the folding and unfolding behavior of tropoelastin and its mutations. Specifically, we derive a discrete mechanical model whose structure is deduced based on a coarse graining approach that allows us to group the amino acids sequence in a smaller number of `equivalent’ masses. Nearest neighbor energy terms are then introduced to reproduce the interaction of such amino acid groups. Nearest and non-nearest neighbor energy terms, inter and intra functional blocks are phenomenologically added in the form of Morse potentials. As we show, the resulting system reproduces important properties of the folding-unfolding mechanical response, including the monotonic and cyclic force-elongation behavior, representing a physiologically important information for elastin. The comparison with the experimental behavior of mutated tropoelastin confirms the predictivity of the model. Statement of significanceClassical approaches to the study of phenomena at the molecular scale such as Molecular Dynamics (MD) represent an incredible tool to unveil mechanical and conformational properties of macromolecules, in particular for biological and medical applications. On the other hand, due to the computational cost, the time and spatial scales are limited. Focusing of the real case of tropoelastin, we propose a new approach based on a careful coarse graining of the system, able to describe the overall properties of the macromolecule and amenable of extension to larger scale effects (protein bundles, protein-protein interactions, cyclic loading). The comparison with tropoelastin behavior, also for mutations, is very promising.

Looking ahead in working memory to guide sequential behaviour

SummaryWorking memory can maintain multiple sensory representations to serve unfolding sequential behaviour, such as while making tea or planning a route. How the human mind juggles internal representations as they become relevant to guide sequential behaviour remains poorly understood. Specifically, while there is good evidence that we can flexibly switch priorities among representations in working memory1, 2, 3, 4, it is unclear how and when dormant memory representations are brought into focus during sequential behaviour. Capitalising on a recently established and temporally precise gaze marker of internal selection5,6, we reveal that the focus in the mind moves to the next-relevant memory representation while behaviour associated with the presently relevant memory representation is still ongoing. Thus, like visual sampling of external objects in the world7, 8, 9, internal visual sampling also ‘looks ahead’ to the next object in memory during sequential behaviour.