CO2-Folded Single-Chain Nanoparticles as Recyclable,Improved Carboxylase Mimics

Emulating the function of natural carboxylases to convert CO₂ under atmospheric condition is a great challenge. Herein we report a class of CO₂-folded single-chain nanoparticles (SCNPs) that can function as recyclable, function-intensified carboxylase mimics. Lewis pair polymers containing bulky Lewis acidic and basic groups as the precursor, can bind CO₂ to drive an intramolecular folding into SCNPs, in which CO₂ as the folded nodes can form gas-bridged bonds. Such bridging linkages highly activate CO₂, which endows the SCNPs with extraordinary catalytic ability that can not only catalyze CO₂-insertion of C(sp³)-H for imitating the natural enzyme's function, it can also act on non-natural carboxylation pathways for C(sp² and sp)-H substrates. The nanocatalysts are of highly catalytic efficiency and recyclability, and can work at room temperature and near ambient CO₂ condition, inspiring a new approach to sustainable C₁ utilization.     

Topological Quantities Determining the Folding/Unfolding Rate of Two-state Folding Proteins

We investigate various topological and energy parameters from the protein native structure and find combinations of some parameters that are well correlated with the rate of folding/unfolding. For folding, the topological quantity that combines the clustering coefficient and the long-range order (or total contact distance/contact order) has a high correlation with the folding rate, expressed as ln k _F , obtained from standard experimental conditions. For unfolding, a combination of the impact of edge removal, obtained from the protein structure, and the stability of the native protein structure, as expressed by the free energy change ΔG, gives a good correlation with unfolding rate, ln k _U .     

Platinum Atoms Dispersed in Single-chain Polymer Nanoparticles

The intramolecular cross-linking of single polymer chains can form single-chain nanoparticles(SCNPs), which have many applications.In this study, styrenic copolymers with pendent triphenylphosphine as the coordination site for platinum ions(Pt(Ⅱ)) and benzocyclobutene as the latent reactive groups are synthesized. Triphenylphosphine groups in the chains can coordinate Pt(Ⅱ) and aid slight single-chain folding in dilute solution. The intramolecular cross-linking caused by the ring-open reaction of benzocyclobutene completes the single-chain collapse and forms stable SCNPs in dilute solution. Pt(Ⅱ) embedded in SCNPs can be further reduced to platinum atoms(Pt(0)). Pt(0) steadily and atomically dispersed in SCNPs exhibits better catalytic properties than normal polymer carried platinum particles do for the reduction of p-nitrophenol to p-aminophenol.     

Platinum(II)‐Crosslinked Single‐Chain Nanoparticles: An Approach towards Recyclable Homogeneous Catalysts

We introduce the synthesis and in‐depth characterization of platinum(II)‐crosslinked single‐chain nanoparticles (Pt^II‐SCNPs) to demonstrate their application as a recyclable homogeneous catalyst. Specifically, a linear precursor copolymer of styrene and 4‐(diphenylphosphino)styrene was synthesized via nitroxide‐mediated polymerization. The triarylphosphine ligand moieties along the backbone allowed for the intramolecular crosslinking of single chains via the addition of [Pt(1,5‐cyclooctadiene)Cl₂] in dilute solution. The successful formation of well‐defined Pt^II‐SCNPs was evidenced by size exclusion chromatography, dynamic light scattering, nuclear magnetic resonance (¹H, ³¹P{¹H}, ¹⁹⁵Pt), and diffusion‐ordered spectroscopy. Finally, the activity of the Pt^II‐SCNPs as homogeneous, yet recyclable catalyst was successfully demonstrated using the example of the amination of allyl alcohol.     

Aromatic Cluster Sensor of Protein Folding: Near‐UV Electronic Circular Dichroism Bands Assigned to Fold Compactness

Both far‐ and near‐UV electronic circular dichroism (ECD) spectra have bands sensitive to thermal unfolding of Trp and Tyr residues containing proteins. Beside spectral changes at 222 nm reporting secondary structural variations (far‐UV range), L_b bands (near‐UV range) are applicable as 3D‐fold sensors of protein's core structure. In this study we show that both L_b(Tyr) and L_b(Trp) ECD bands could be used as sensors of fold compactness. ECD is a relative method and thus requires NMR referencing and cross‐validation, also provided here. The ensemble of 204 ECD spectra of Trp‐cage miniproteins is analysed as a training set for “calibrating” Trp?Tyr folded systems of known NMR structure. While in the far‐UV ECD spectra changes are linear as a function of the temperature, near‐UV ECD data indicate a non‐linear and thus, cooperative unfolding mechanism of these proteins. Ensemble of ECD spectra deconvoluted gives both conformational weights and insight to a protein folding?unfolding mechanism. We found that the L_b²⁹³ band is reporting on the 3D‐structure compactness. In addition, the pure near‐UV ECD spectrum of the unfolded state is described here for the first time. Thus, ECD folding information now validated can be applied with confidence in a large thermal window (5≤T≤85 °C) compared to NMR for studying the unfolding of Trp?Tyr residue pairs. In conclusion, folding propensities of important proteins (RNA polymerase II, ubiquitin protein ligase, tryptase‐inhibitor etc.) can now be analysed with higher confidence.     

A Single‐Molecule Perspective on the Role of Solvent Hydrogen Bonds in Protein Folding and Chemical Reactions

We present an array of force spectroscopy experiments that aim to identify the role of solvent hydrogen bonds in protein folding and chemical reactions at the single‐molecule level. In our experiments we control the strength of hydrogen bonds in the solvent environment by substituting water (H₂O) with deuterium oxide (D₂O). Using a combination of force protocols, we demonstrate that protein unfolding, protein collapse, protein folding and a chemical reaction are affected in different ways by substituting H₂O with D₂O. We find that D₂O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D₂O is a worse solvent than H₂O for the protein I27, in direct contrast with the behaviour of simple hydrocarbons. We measure the effect of substituting H₂O with D₂O on the force dependent rate of reduction of a disulphide bond engineered within a single protein. Altogether, these experiments provide new information on the nature of the underlying interactions in protein folding and chemical reactions and demonstrate the power of single‐molecule techniques to identify the changes induced by a small change in hydrogen bond strength.     

Folding behaviors of apocytochrome b5 and its mutants: Insights from high temperature molecular dynamics simulations

Apocytochrome b₅ (apocyt b₅) with heme removal from the heme-binding core 1, and its mutants with amino acid replaced in the hydrophobic core 2, namely apocyt b₅ Y7P, P81A and H15R/S20E, have been subjected to molecular dynamics (MD) simulation at high temperature (500 K) for elucidating their folding behaviors. The early events upon thermal induced unfolding were found to be in good agreement with available experimental results, and the lowest stability of Y7P was predicted among the four apoproteins. The influences of these key residues on protein folding behavior were compared directly at an atomic level. At the same time, the influences of non-native interactions of hydrogen bonds and salt-bridges on protein stabilities were analyzed in detail. The insights from current MD simulations are valuable for understanding the apoprotein folding and the holoprotein formation in terms of heme proteins.     

Do Valine Side Chains Have an Influence on the Folding Behavior of β‐Substituted β‐Peptides?

The influence of valine side chains on the folding/unfolding equilibrium and, in particular, on the 3₁₄‐helical propensity of β³‐peptides were investigated by means of molecular‐dynamics (MD) simulation. To that end, the valine side chains in two different β³‐peptides were substituted by leucine side chains. The resulting four peptides, of which three have never been synthesized, were simulated for 150 to 200 ns at 298 and 340 K, starting from a fully extended conformation. The simulation trajectories obtained were compared with respect to structural preferences and folding behavior. All four peptides showed a similar folding behavior and were found to predominantly adopt 3₁₄‐helical conformations, irrespective of the presence of valine side chains. No other well‐defined conformation was observed at significant population in any of the simulations. Our results imply that β³‐peptides show a structural preference for 3₁₄‐helices independent of the branching nature of the side chains, in contrast to what has been previously proposed on the basis of circular‐dichroism (CD) measurements.     

Helical Folding Competing with Unfolded Aggregation in Phenylene Ethynylene Foldamers

The folding and aggregation behavior of a pair of oligo(phenylene ethynylene) (OPE) foldamers are investigated by means of UV/Vis absorption and circular dichroism spectroscopy. With identical OPE backbones, two foldamers, 1 with alkyl side groups and 2 with triethylene glycol side chains, manifest similar helical conformations in solutions in n‐hexane and methanol, respectively. However, disparate and competing folding and aggregation processes are observed in alternative solvents. In cyclohexane, oligomer 1 initially adopts the helical conformation, but the self‐aggregation of unfolded chains, as a minor component, gradually drives the folding–unfolding transition eventually to the unfolded aggregate state completely. In contrast, in aqueous solution (CH₃OH/H₂O) both folded and unfolded oligomer 2 appear to undergo self‐association; aggregates of the folded chains are thermodynamically more stable. In solutions with a high H₂O content, self‐aggregation among unfolded oligomers is kinetically favored; these oligomers very slowly transform into aggregates of helical structures with greater thermodynamic stability. The folded–unfolded conformational switch thus takes place with the free (nonaggregated) molecules, and the very slow folding transition is due to the low concentration of molecularly dispersed oligomers.     

Structural Insights into the Trp‐Cage Folding Intermediate Formation

The 20 residue long Trp‐cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the ¹⁵N and ¹³C/¹⁵N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible—fully folded, unfolded and intermediate—fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH^F–I, T_m^F–I, ΔC_p^F–I and ΔH^I–U, T_m^I–U, ΔC_p^I–U) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp‐cage distinct intermediates evolve: a fast‐exchanging intermediate is present under neutral conditions, whereas a slow‐exchanging intermediate‐pair emerges at acidic pH. The fast‐exchanging intermediate has a native‐like structure with a short α‐helix in the G¹¹–G¹⁵ segment, whereas the slow‐exchanging intermediate‐pair presents elevated dynamics, with no detectable native‐like residue contacts in which the G¹¹? P¹² peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.     

Evaluation of thermodynamic properties of irreversible protein thermal unfolding measured by DSC

Summary We assessed the applicability of the extrapolation procedure at infinite scanning rate to differential scanning calorimetry (DSC) data related to irreversible protein unfolding. To this aim, an array of DSC curves have been simulated on the basis of the Lumry-Eyring model N↔U→F. The results obtained confirmed that when the apparent equilibrium constant K_{app (T=T1/2)} is lower than 3, the application of the extrapolation procedure provides accurate thermodynamic parameters. Although this procedure applies only to monomeric proteins for which the Lumry-Eyring model is a reasonable approximation, it will hopefully contribute to increase the potential of DSC in obtaining reliable thermodynamic information regarding the folding/unfolding equilibrium.     

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well‐defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15‐mer vascular endothelial growth factor mimicking α‐helical peptide (QK_L10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK_L10A H_α chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high‐resolution QK_L10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding–unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding–unfolding picture for the small peptide QK_L10A compatible with the nucleation–propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein–protein interactions.     

Direct probing of the folding/unfolding event of bovine hemoglobin at montmorillonite clay modified electrode by adsorptive-transfer voltammetry

An efficient way was proposed for probing the folding/unfolding event of bovine hemoglobin (Hb) through adsorptive-transfer voltammetry. Hb molecules in native and pre-unfolded in different urea conditions for 23 h were adsorbed onto the montmorillonite clay modified glassy carbon electrode (Hb/clay/GCE and uHb/clay/GCE, respectively). Cyclic voltammograms of Hb/clay/GCE and uHb/clay/GCE showed that the unfolding of Hb caused great change in the direct electron transfer between the heme irons within Hb and electrode surface, which was facilitated on clay film. From the amount of the electroactive Hb (W_Hbe) and the adsorbed Hb (Γ) on clay per unit mass, the minimal electroactive portion (MEP) of the adsorbed Hb was calculated to assess the unfolding state of Hb. With the increase of urea concentration, MEP showed a sigmoid curve. Thermodynamic parameters related to the unfolding event of Hb were also obtained based on the linear free energy model (LEM), including the free energy of folding in water (ΔG_U^water), the slope of the Santoro-Bolen equation (m), and the urea concentration required in for achieving half of the total change (S_m) in the unfolding curves. This work gave the first try for investigating protein unfolding at nano-materials modified electrode using adsorptive-transfer voltammetry, which improved the sensitivity of analysis and avoided the disadvantages involved in the existing electrochemical methods for protein unfolding. The proposed method will benefit the electrochemical studies of protein.     

X-Ray-triggered Carbon Monoxide and Manganese Dioxide Generation based on Scintillating Nanoparticles for Cascade Cancer Radiosensitization

Carbon monoxide (CO) is an endogenous signaling molecule with broad therapeutic effects. Here, a multifunctional X-ray-triggered carbon monoxide (CO) and manganese dioxide (MnO₂) generation nanoplatform based on metal carbonyl and scintillating nanoparticles (SCNPs) is reported. Attributed to the radioluminescent characteristic of SCNPs, UV-responsive Mn₂(CO)₁₀ is not only indirectly activated to release CO by X-ray but can also be degraded into MnO₂. A high dose of CO can be used as a glycolytic inhibitor for tumor suppression; it will also sensitize tumor cells to radiotherapy. Meanwhile MnO₂, as the photolytic byproduct of Mn₂(CO)₁₀, has both glutathione (GSH) depletion and Fenton-like Mn²⁺ delivery properties to produce highly toxic hydroxyl radical (⋅OH) in tumors. Thus, this strategy can realize X-ray-activated CO release, GSH depletion, and ⋅OH generation for cascade cancer radiosensitization. Furthermore, X-ray-activated Mn²⁺ in vivo demonstrates an MRI contrast effect, making it a potential theranostic nanoplatform.     

Thermal stability and folding kinetics analysis of disordered protein,securin

Lacking a stable tertiary structure, intrinsically disordered proteins (IDPs) possess particular functions in cell regulation, signaling, and controlling pathways. The study of their unique structural features, thermal stabilities, and folding kinetics is intriguing. In this study, an identified IDP, securin, was used as a model protein. By using a quasi-static five-step (on-path) folding process, the function of securin was restored and analyzed by isothermal titration calorimetry. Fluorescence spectroscopy and particle size analysis indicated that securin possessed a compact hydrophobic core and particle size. The glass transition of securin was characterized using differential scanning microcalorimetry. Furthermore, the folding/unfolding rates (k_obs) of securin were undetectable, implying that the folding/unfolding rate is very fast and that the conformation of securin is sensitive to solvent environmental change. Therefore, securin may fold properly under specific physiological conditions. In summary, the thermal glass transition behavior and undetectable k_obs of folding/unfolding reactions may be two of the indices of IDP.     

Metal‐Folded Single‐Chain Nanoparticle: Nanoclusters and Self‐Assembled Reduction‐Responsive Sub‐5‐nm Discrete Subdomains

Easy access to discrete nanoclusters in metal‐folded single‐chain nanoparticles (metal‐SCNPs) and independent ultrafine sudomains in the assemblies via coordination‐driven self‐assembly of hydrophilic copolymer containing 9% imidazole groups is reported herein. ¹H NMR, dynamic light scattering, and NMR diffusion‐ordered spectroscopy results demonstrate self‐assembly into metal‐SCNPs (>70% imidazole‐units folded) by neutralization in the presence of Cu(II) in water to pH 4.6. Further neutralization induces self‐assembly of metal‐SCNPs (pH 4.6–5.0) and shrinkage (pH 5.0–5.6), with concurrent restraining residual imidazole motifs and hydrophilic segment, which organized into constant nanoparticles over pH 5.6–7.5. Atomic force microscopy results evidence discrete 1.2 nm nanoclusters and sub‐5‐nm subdomains in metal‐SCNP and assembled nanoparticle. Reduction of metal center using sodium ascorbate induces structural rearrangement to one order lower than the precursor. Enzyme mimic catalysis required media‐tunable discrete ultrafine interiors in metal‐SCNPs and assemblies have hence been achieved.     

Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Instant Locking of Molecular Ordering in Liquid Crystal Elastomers by Oxygen‐Mediated Thiol–Acrylate Click Reactions

Liquid crystal elastomers (LCEs) with intrinsic anisotropic strains are reversible shape‐memory polymers of interest in sensor, actuator, and soft robotics applications. Rapid gelation of LCEs is required to fix molecular ordering within the elastomer network, which is essential for directed shape transformation. A highly efficient photo‐cross‐linking chemistry, based on two‐step oxygen‐mediated thiol–acrylate click reactions, allows for nearly instant gelation of the main‐chain LCE network upon exposure to UV light. Molecular orientation from the pre‐aligned liquid crystal oligomers can be faithfully transferred to the LCE films, allowing for preprogrammed shape morphing from two to three dimensions by origami‐ (folding‐only) and kirigami‐like (folding with cutting) mechanisms. The new LCE chemistry also enables widely tunable physical properties, including nematic‐to‐ isotropic phase‐transition temperatures (T_N‐I), glassy transition temperatures (T_g), and mechanical strains, without disrupting the LC ordering.     

Oligopeptide Helical Conformations Control Gold Nanoparticle Cross-Linking

Peptide sequences functionalized with primary amines at the N- and C-terminus are able to induce the aggregation of gold nanoparticles in ethanol as a consequence of their folding into a helical conformation. Random coil peptides are unable to induce such an aggregation process. Aggregation can be monitored spectrophotometrically by following the shift of the surface plasmon resonance (SPR) band of the nanoparticles and is confirmed by transmission electron microscopy and dynamic light scattering analyses. Partial denaturation of the peptides results in diminished cross-linking ability. The helicity parameter θ₂₂₂/θ₂₀₈ correlates fairly well with the shift of the SPR band to longer wavelengths, supporting the relationship between the amount of helical content of a peptide sequence and its ability to induce aggregation.