Self-association Behavior Research Articles

A scaling relationship between thermodynamic and hydrodynamic interactions in protein solutions

Weak protein interactions are associated with a broad array of biological functions and are often implicated in molecular dysfunction accompanying human disease. In addition, these interactions are a critical determinant in the effective manufacturing, stability, and administration of biotherapeutic proteins. Despite their prominence, much remains unknown about how molecular attributes influence the hydrodynamic and thermodynamic contributions to the overall interaction mechanism. To systematically probe these contributions, we have evaluated self-interaction in a diverse set of proteins that demonstrate a broad range of behaviors from attractive to repulsive. Analysis of the composite trending in the data provides a convenient interconversion among interaction parameters measured from the concentration dependence of the molecular weight, diffusion coefficient, and sedimentation coefficient, as well as insight into the relationship between thermodynamic and hydrodynamic interactions. We find relatively good agreement between our data and a model for interacting hard spheres in the range of weak self-association. In addition, we propose an empirically derived, general scaling relationship applicable across a broad range of self-association and repulsive behaviors.

Concentration-Dependent Diffusion of Monoclonal Antibodies: Underlying Mechanisms of Anomalous Diffusion.

The development, storage, transport, and subcutaneous delivery of highly concentrated monoclonal antibody formulations pose significant challenges due to the high solution viscosity and low diffusion of the antibody molecules in crowded environments. These issues often stem from the self-associating behavior of the antibody molecules, potentially leading to aggregation. In this work, we used a dissipative particle dynamics-based coarse-grained model to investigate the diffusion behavior of IgG1 antibody molecules in aqueous solutions with 15 and 32 mM NaCl and antibody concentrations ranging from 10 to 400 mg/mL. We determined the coarse-grained interaction parameters by matching the calculated structure factor with the computational and experimental data from the literature. Our results indicate Fickian diffusion for antibody concentrations of 10 and 25 mg/mL and anomalous diffusion for concentrations exceeding 50 mg/mL. The anomalous diffusion was observed for ∼0.33 to 0.4 μs, followed by Fickian diffusion for all antibody concentrations. We observed a strong linear correlation between the diffusion behavior of the antibody molecules (diffusion coefficient D and anomalous diffusion exponent α) and the amount of aggregates present in the solution and between the amount of aggregates and the Coulomb interaction energy. The investigation of underlying mechanisms for anomalous diffusion revealed that in crowded environments at high antibody concentrations, the attractive interaction between electrostatically complementary regions of the antibody molecules could further bring the neighboring molecules closer to one another, ultimately resulting in aggregate formation. Further, the Coulomb attraction can continue to draw more molecules together, forming larger aggregates.

Phase behavior of patchy colloids confined in patchy porous media.

A simple model for functionalized disordered porous media is proposed and the effects of confinement on self-association, percolation and phase behavior of a fluid of patchy particles are studied. The media are formed by randomly distributed hard-sphere obstacles fixed in space and decorated by a certain number of off-center square-well sites. The properties of the fluid of patchy particles, represented by the fluid of hard spheres each bearing a set of the off-center square-well sites, are studied using an appropriate combination of the scaled particle theory for the porous media, Wertheim's thermodynamic perturbation theory, and Flory-Stockmayer theory. To assess the accuracy of the theory a set of computer simulations have been performed. In general, predictions of the theory appeared to be in good agreement with the computer simulation results. Confinement and competition between the formation of bonds connecting the fluid particles, and connecting fluid particles and obstacles of the matrix, gave rise to a re-entrant phase behavior with three critical points and two separate regions of the liquid-gas phase coexistence.

Tailoring the Interplay between Two Monomers in the Properties of Degradable Polyesters Synthesized via Ring-Opening Alternating Copolymerization.

Ring-opening alternating copolymerization (ROAC) of cyclic anhydrides and epoxides has emerged as a powerful strategy to produce degradable polyesters with a diverse array of structures from the combination of two distinct building blocks. In this work, we exploited the organocatalytic ROAC of cyclic anhydrides and a functional epoxide, t-butyl glycidoxy acetate, followed by acidic deprotection to access degradable polyesters with carboxylic acid pendants. To study the interplay between monomers, diglycolic anhydride and glutaric anhydride were used as cyclic anhydrides to prepare two polyesters. In particular, the effects of the oxygen heteroatom in the cyclic anhydrides on the properties of the carboxylic acid-containing polyesters were investigated. The introduction of the oxygen heteroatom into the cyclic anhydrides significantly influenced their thermal properties and pH-dependent self-association behavior in an aqueous solution. Furthermore, molecular dynamics simulations elucidate that the number and type of hydrogen bonds play a crucial role in the self-association behavior between the polymers both in the solution and bulk states. The findings of this study highlight the importance of the interplay between monomers in the design of functional polyesters with tunable properties.

In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Doublet Open‐Shell Graphene Fragments

Recent advances in neutral delocalized radical species based on polycyclic aromatic hydrocarbons with fused hexagonal rings, herein defined as doublet open-shell graphene fragments, are summarized in this review. A few simple yet useful theoretical approaches for structural analysis and molecular design were introduced at first. Then, based on the number of fused hexagonal rings, molecular systems with different size, symmetry and edge structure were discussed with emphasis on those isolated in the crystalline form. Their unique self-association behavior, chemical reactivity and physical properties were summarized and discussed, and insights on their functions and potential applications were provided.

Augmented Self-Association by Electrostatic Forces in Thienopyrrole-Fused Thiadiazoles that Contain an Ester instead of an Ether Linker.

During the self-assembly of π-conjugated molecules, linkers and substituents can potentially add supportive noncovalent intermolecular interactions to π-stacking interactions. Here, we report the self-assembly behavior of thienopyrrole-fused thiadiazole (TPT) fluorescent dyes that possess ester or ether linkers and dodecyloxy side chains in solution and the condensed phase. A comparison of the self-association behavior of the ester- and ether-bridged compounds in solution using detailed UV-vis, fluorescence, and NMR spectroscopic studies revealed that the subtle replacement of the ether linkers by ester linkers leads to a distinct increase in the association constant (ca. 3-4 fold) and the enthalpic contribution (ca. 3 kcal mol-1 ). Theoretical calculations suggest that the ester linkers, which are in close proximity to one another due to the π-stacking interactions, induce attractive electrostatic forces and augment self-association. The self-assembly of TPT dyes into well-defined 1D clusters with high aspect ratios was observed, and their morphologies and crystallinity were investigated using SEM and X-ray diffraction analyses. TPTs with ester linkers exhibit a columnar liquid crystalline mesophase in the condensed phase.

β-lactoglobulin conformation influences its interaction with caffeine

Caffeine is a molecule with bioactive properties related to increased mental performance. Its use in food products is limited by its bitterness and self-association behavior. The complex formed by β-lactoglobulin and caffeine was characterized by fluorescence spectroscopy, isothermal titration calorimetry, and molecular docking. Fluorescence spectroscopy revealed that β-lactoglobulin interacted with caffeine to form a 1:1 stoichiometric complex. Isothermal titration calorimetry characterization of β-lactoglobulin-caffeine binding revealed a different stoichiometry (1:3). Molecular docking analysis confirmed the information obtained by the two thermodynamic evaluation techniques, showing that the best interaction occurred in the hydrophobic cavity of β-lactoglobulin. The effect of protein conformation was also evaluated by fluorescence spectroscopy. The complex stoichiometry and binding constants were from the same order as those obtained for native β-lactoglobulin. However, in contrast to the complex formation with native protein, the flexibility increase observed in the thermal unfolded protein promoted a great temperature effect on the standard enthalpy and entropy changes of interaction, indicating the presence of a balance between hydrophobic and hydrophilic forces.

Polyelectrolyte complex induced Stimuli-responsive Self-association and reinforcement of interpenetrated Poly(acrylamide-co-vinyl acetate)/alginate particles for fossil energy recovery

Stimuli-responsive poly(acrylamide-co-vinyl acetate)/alginate-based hydrogel particle (AIPNG particle) are obtained by free-radical polymerization of acrylamide monomer and vinyl acetate monomer which interpenetrates with branched polyethyleneimine (B-PEI)/alginate-based polyelectrolyte complex (PEC). The AIPNG particle is developed for reservoir conformance control in Ca-rich reservoirs. The AIPNG particle can swell 13 to 25 times its initial volume, and the equilibrium swelling ratio is dependent on brine type and concentration. The swelling and elastic modulus of the AIPNG particles are strongly influenced by salt in the aqueous solution. The release rate of B-PEI from B-PEI/alginate PEC structure, combining with the swelling ratio, the environmental temperature, and Ca2+ concentration, determines the self-association time and rheological strength of the AIPNG gel. Preparation pH influences the protonation degree of B-PEI and subsequently affects the self-association behavior by delaying the intermolecular transamidation reaction between poly(AM-co-VA) acrylamide pendant groups and PEI nitrogens. With the component adjustments, the self-association starting time can be controlled between 2 and 16 h depending on temperature and swelling ratio. Ca2+, supported from environmental brine, can influence the self-association time and help reinforce the re-formed bulk gel by forming Ca-alginate bonds and creating a hard Ca-shell on the gel surface. Field trial results in Zhidan oilfield demonstrated the potential of AIPNG particle products in high-salinity/middle-temperature reservoirs which provided a new way for maintaining the oil production and reducing water-cut in the mature oilfields.

Understanding the Effects of Phosphorylation on the Structural Dynamics of EWS‐FLI1 and its Self‐Associative Behavior

Ewing sarcoma is a malignant tumor that occurs in bones or soft tissues, such as cartilage or nerves. Ewing sarcoma is a rare cancer occurring with a global frequency of approximately 1.5 cases per million children and adolescents. Ewing tumors are treatable if diagnosed early, but their aggressiveness leads to metastasis or relapse, upon which the survival rate is reduced to less than 30 percent. Development of targeted therapies are urgently needed since the current standard of care using repurposed adult cancer therapeutics are extremely toxic leaving juvenile patients with lifelong debilities and reduced lifespan. Ewing sarcoma is characterized by a chromosomal translocation, most commonly t(11;22)(q24;q12), which joins the low-complexity domain of the RNA-binding protein Ewing sarcoma breakpoint region 1 (EWSR1) with DNA binding domain of the transcription factor, Friend Leukemia Integration 1 (FLI1). The resultant oncogenic fusion protein, Ewing sarcoma-Friend Leukemia Integration 1 (EWS-FLI1) is considered an intrinsically disordered protein (IDP), a class of proteins that are functional, yet lack stable secondary or tertiary structures. The structural dynamics and functions of IDPs are frequently associated with post-translational modification (PTM) sites, which serve to regulate biological functions such as biomolecular interactions or sub-cellular localization. By analogy with Fused in Sarcoma (FUS), a closely related family member of EWSR1, we have identified 8 DNA PK (S/TQ) target sites in the EWS portion of EWS-FLI1. Because protein phosphorylation is used as a mechanism to control many life critical processes, we are hypothesizing that phosphorylation of EWS-FLI1 is an important modulator for its activity. Using an in vitro phosphorylation assay, we have shown that DNA PK phosphorylates several of these sites. We are now interested in observing the structural and biophysical impacts of phosphorylation on EWS-FLI1 including the potential modulation of its self-associative function. We will use NMR and mass spectroscopy to identify the phosphorylation sites, determine the frequency of modification and assess the impact of this PTM on the structure of EWS-FLI1. Finally, using S/T to E as phosphomimetic mutations we will examine the impact of phosphorylation on the self-associative function of EWS-FLI1 using fluorescence-based aggregation assay. If successful, this will help us to understand the function of EWS-FLI1's self-associative behavior and identify novel therapeutic vulnerabilities.

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. Here we report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid–liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by these brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and aggregation. We suggest that this variability among ubiquilins along the continuum from liquid-like to solid informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in neurodegenerative diseases.

Linker histone defines structure and self-association behaviour of the 177\xa0bp human chromatosome

Linker histones play essential roles in the regulation and maintenance of the dynamic chromatin structure of higher eukaryotes. The influence of human histone H1.0 on the nucleosome structure and biophysical properties of the resulting chromatosome were investigated and compared with the 177-bp nucleosome using Cryo-EM and SAXS. The 4.5 Å Cryo-EM chromatosome structure showed that the linker histone binds at the nucleosome dyad interacting with both linker DNA arms but in a tilted manner leaning towards one of the linker sides. The chromatosome is laterally compacted and rigid in the dyad and linker DNA area, in comparison with the nucleosome where linker DNA region is more flexible and displays structural variability. In solution, the chromatosomes appear slightly larger than the nucleosomes, with the volume increase compared to the bound linker histone, according to solution SAXS measurements. SAXS X-ray diffraction characterisation of Mg-precipitated samples showed that the different shapes of the 177 chromatosome enabled the formation of a highly ordered lamello-columnar phase when precipitated by Mg2+, indicating the influence of linker histone on the nucleosome stacking. The biological significance of linker histone, therefore, may be affected by the change in the polyelectrolyte and DNA conformation properties of the chromatosomes, in comparison to nucleosomes.

Complexation of 2,7-diazapyrene with boron for structural and electronic tuning.

Complexation of 1,3,6,8-tetra(2-hydroxyphenyl)-2,7-diazapyrene with boron precursors provided the tetracoordinate diazapyrene boron complexes as separable anti- and syn-isomers. The structural difference of these isomers induces unique properties such as self-association behaviour of the syn-isomer and isomerisation of the anti-isomer in the solution and solid states.

Extremely Amyloidogenic Single-Chain Analogues of Insulin's H-Fragment: Structural Adaptability of an Amyloid Stretch.

Relatively short amino acid sequences often play a pivotal role in triggering protein aggregation leading to the formation of amyloid fibrils. In the case of insulin, various regions of A- and B-chains have been implicated as the most relevant to the protein’s amyloidogenicity. Here, we focus on the highly amyloidogenic H-fragment of insulin comprising the disulfide-bonded N-terminal parts of both chains. Analysis of the aggregation behavior of single-chain peptide derivatives of the H-fragment suggests that the A-chain’s part initiates the aggregation process while the disulfide-tethered B-chain reluctantly adapts to amyloid structure. Merging of both A- and B-parts into single-chain continuous peptides (A–B and B–A) results in extreme amyloidogenicity exceeding that of the double-chain H-fragment as reflected by almost instantaneous de novo fibrillization. Amyloid fibrils of A–B and B–A present distinct morphological and infrared traits and do not cross-seed insulin. Our study suggests that the N-terminal part of insulin’s A-chain containing the intact Cys6–Cys11 intrachain disulfide bond may constitute insulin’s major amyloid stretch which, through its bent conformation, enforces a parallel in-register alignment of β-strands. Comparison of the self-association behavior of H, A–B, and B–A peptides suggests that A-chain’s N-terminal amyloid stretch is very versatile and adaptive to various structural contexts.

Synthesis of Amphiphilic Statistical Copolymers Bearing Methoxyethyl and Phosphorylcholine Groups and Their Self-Association Behavior in Water.

Biocompatible amphiphilic statistical copolymers P(MEA/MPCm) composed of 2-methoxyethyl acrylate (MEA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) were prepared with three different mol% of the hydrophilic unit MPC (m = 6, 12 and 46 mol%). The monomer reactivity ratios of MEA (rMEA) and MPC (rMPC) were 0.53 and 2.21, respectively. The rMEA × rMPC value of 1.17 demonstrated that statistical copolymerization was successful. P(MEA/MPC12) and P(MEA/MPC46) copolymers did not undergo aggregation in water, whereas the P(MEA/MPC6) copolymer formed micelles in water with a hydrodynamic radius (Rh) of 96.9 nm and a critical aggregation concentration, which was determined using pyrene fluorescence, at 0.0082 g/L. The restricted motion of the protons in the hydrophobic MEA units in the micelles’ cores provided additional evidence of self-association in P(MEA/MPC6).

Helical Nanographenes Embedded with Contiguous Azulene Units.

The azulene moiety, composed of contiguous pentagonal and heptagonal rings, is a structural defect that alters the electronic, magnetic, and structural properties of graphenes and graphene nanoribbons. However, nanographenes embedded with an azulene cluster have not been widely investigated because these compounds are difficult to synthesize in their pure form. Herein, azulene-embedded nanographenes bearing a unique cove-type edge were synthesized by a novel synthetic protocol. Experimental and theoretical investigations revealed that this cove edge imparts stable helical chirality, unlike normal cove edges. The in-solution self-association behavior and the structural, electronic, and electrochemical properties were also described in detail.

Multiscale Coarse-Grained Approach to Investigate Self-Association of Antibodies

Self-association of therapeutic monoclonal antibodies (mabs) are thought to modulate the undesirably high viscosity observed in their concentrated solutions. Computational prediction of such a self-association behavior is advantageous early during mab drug candidate selection when material availability is limited. Here, we present a coarse-grained (CG) simulation method that enables microsecond molecular dynamics simulations of full-length antibodies at high concentrations. The proposed approach differs from others in two ways: first, charges are assigned to CG beads in an effort to reproduce molecular multipole moments and charge asymmetry of full-length antibodies instead of only localized charges. This leads to great improvements in the agreement between CG and all-atom electrostatic fields. Second, the distinctive hydrophobic character of each antibody is incorporated through empirical adjustments to the short-range van der Waals terms dictated by cosolvent all-atom molecular dynamics simulations of antibody variable regions. CG simulations performed on a set of 15 different mabs reveal that diffusion coefficients in crowded environments are markedly impacted by intermolecular interactions. Diffusion coefficients computed from the simulations are in correlation with experimentally measured observables, including viscosities at a high concentration. Further, we show that the evaluation of electrostatic and hydrophobic characters of the mabs is useful in predicting the nonuniform effect of salt on the viscosity of mab solutions. This CG modeling approach is particularly applicable as a material-free screening tool for selecting antibody candidates with desirable viscosity properties.

Rapid self-association of highly amyloidogenic H-fragments of insulin: Experiment and molecular dynamics simulations

The so-called ‘H-fragment’ of insulin is an extremely amyloidogenic double chain peptide consisting of the N-terminal parts of A-chain and B-chain linked by a disulfide bond between Cys-7A and Cys-7B. Here, we conduct a detailed investigation of the self-association behavior of H-fragment monomers into amyloid-like fibrils using kinetic assays, infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Unlike the intact predominantly α-helical insulin, H-fragment remains in a disordered state in aqueous solutions. Its aggregation accelerates with acidification of the environment leading, at pH 1.9, to the formation of thin and structurally homogenous fibrils with the infrared features typical for parallel β-sheet conformation. According to time-lapse AFM morphological analysis both secondary nucleation and fragmentation are involved in later stages of H-fibrils' self-assembly. Based on the low nucleation order (two) obtained from the global fitting of kinetic data, realistic all-atom MD simulations of pairs of interacting H-fragment monomers were subsequently carried out. The molecular self-association scenario emerging from these simulations implicates the intrinsic conformational instability of H-monomer in its tendency to aggregate and form intermolecular β-sheet structure. Our findings provide the new mechanistic context for studies of insulin misfolding and aggregation.

Tautomerism and Self-Association in the Solution of New Pinene-Bipyridine and Pinene-Phenanthroline Derivatives.

Two novel pinene-type ligands have been synthesized and their tautomeric and self-associating behavior studied in solution and in the solid state. The first ligand, an acetylated derivative of 5,6-pinene-bipyridine, displays keto-enol tautomerism in solution. This tautomeric equilibrium was studied by NMR and UV-Vis spectroscopy in various solvents, and the results were compared with the ones obtained through DFT calculations. The second ligand was obtained by an unusual oxidation of the phenanthroline unit of a pinene-phenanthroline derivative. This compound exists as a single tautomer in solution and aggregates both in solution (as observed by NMR) and in the solid state through H-bonding as observed by X-ray structure determination and confirmed by DFT studies.

Micellization properties of antihistaminic drug diphenhydramine.HCl in aqueous electrolytic solution: Conductometric and spectroscopic studies

The empathetic knowledge about the drug-electrolyte interactions is imperative in understanding the pharmacology and pharmacokinetics of drug. Therefore in the present paper, the effect of aqueous electrolytic solutions on the micellization behavior of an antihistaminic drug diphenhydramine.HCl (DPH) has been extensively examined through conductometric, fluorometric and spectrophotometric studies. The effect of various electrolytes occurring in the body fluid on self-association behavior of DPH was evaluated in terms of critical micelle concentration (CMC) obtained by conductivity and spectroscopic data over the temperature range (298.15–313.15) K with the regular interval of 5 K. The so obtained CMC data is used to calculate standard Gibbs free energy change of micellization (ΔGmo), entropy change of micellization (ΔGmo), and enthalpy change of micellization (ΔHmo), for the aforesaid drug in the presence of various electrolytes. The standard thermodynamic parameters of micellization were used to examine various solute-solvent interactions existing in drug-electrolyte-water system under discussion. Various thermodynamic parameters viz. ΔGmo, ΔHmo, and ΔGmo found to support the fact that electrolytes favor the association behavior of the DPH, which is further validated by pseudo-phase model.