Kinetics Of Dimerization Research Articles

Exact solutions of reversible dimerization and trimerization kinetics

Exact solutions of reversible dimerization and trimerization kinetics

Kinetics of Polyampholyte Dimerization: Influence of Charge Sequences

Polyampholytes (PAs) exhibit complex behaviors in various environments influenced by their charge distribution. This study focuses on the kinetics of dimerization of PAs, aiming to elucidate the underlying mechanisms and clarify relevant characteristics of the charge sequence. We focus on PAs with non-zero net charges, employing molecular dynamics simulations and theoretical analyses to examine how charge sequences influence the rates of dimer formation and dissociation. Our findings reveal that the charge sequence of tails and the blockiness of the minority charge group markedly influence the kinetics of dimerization: large blockiness and tails with a high number of majority-type charges slow down the dissociation of dimers. Additionally, the presence of an extended (central) block of the majority charge promotes structural diversity. Within dimer states, blocks alternate between intra- and inter-chain contacts. The duration times in the dimer states are significantly longer than the typical dwell times of block inter-contacts, with a notable extension when multiple blocks are engaged. Intrinsically disordered proteins (IDPs) play crucial roles in cellular functions, primarily due to their ability to undergo rapid conformational changes and form transient complexes. These properties largely depend on the sequence of charged residues. We provide insights into the fundamental principles governing the structural and dynamical properties of polyampholytic IDP, emphasizing the importance of sequence-specific effects on both aggregation and dissociation.

Temperature‐Programmed Reflection Absorption Infrared Spectroscopy: A Methods Review

AbstractA method is proposed to measure the kinetics of elementary‐step reactions using reflection‐absorption infrared spectroscopy (RAIRS) by slowly ramping the sample temperature while continually collecting the interferograms, where the interferograms are post‐processed to provide temperature‐dependent spectra. Heating rates of ~10−2 K/s allow spectra to be collected so that the temperature is essentially constant for each spectrum. This strategy can allow short‐lived intermediates to be observed. The data are fit to analytical formulae similar to those used for conventional temperature‐programmed desorption and the two approaches are complementary. The method is illustrated by measuring the activation energy for the ordering of crystalline thin films and for multilayer desorption. The utility of the approach for measuring surface‐chemical processes is illustrated for the dimerization kinetics of a prochiral reactant, methyl pyruvate (CH3CCO2CH3), on Pd(111), where the monomer has been found to be more reactive than the dimer, and the decarbonylation steps of furfural (C4H3O ⋅ CHO), a product of the treatment of biomass. The results are in good agreement with cases in which there are other methods for measuring the reaction rates. The approach is also useful for establishing how accurate the results of theoretical simulations are for elementary surface reactions.

In-situ Cu2O/Cu interfaces generation on Cu2O nanocubes toward enhanced C2+ selectivity

The electro-reduction of CO2 to C2+ products has received a lot of attention because it provides a pathway to obtain feedstocks and fuels by renewable electricity. It is well known that Cu-based catalysts are selective for C2+ products, although the catalytic mechanism of its surface structure is not fully understood. Therefore, we procured Cu2O nanocubes via a wet chemical reduction approach, and then Cu2O/Cu catalyst was in-situ generated during the electro-catalytic CO2 reduction reaction (CO2RR). The electrochemical properties of Cu2O/Cu catalyst are better than those of Cu catalyst. In particular, the selectivity of Cu2O/Cu catalyst for C2+ products is 55.9 %, which is approximately 67 % higher than that of Cu catalyst at the optimal catalytic voltage of –1.1 V versus reversible hydrogen electrode (RHE). In addition, the stability of Cu2O/Cu catalyst is up to 30 h, and the catalytic current density is significantly higher than that of Cu catalyst. Using in-situ Raman spectroscopy, the signal peaks of transition state intermediates (*CO–Cu and CO) of Cu2O/Cu catalyst are stronger than those of Cu catalyst during CO2RR. Density function theory (DFT) calculations suggest that the good electrochemical performance is associated with the Cu2O/Cu interfaces, which significantly improves the thermodynamics and kinetics of CO2 activation and CO dimerization. This study provides a simple approach for improving catalytic performance and will advance the understanding of the role of the Cu2O/Cu interfaces in CO2RR.

Protein-protein association properties of human βB2-crystallins.

Protein-protein association events are involved in many physiological and pathological processes. Cataract disease is a pathology that manifests protein aggregation of crystallins. β-Crystallins are present in a high proportion in the eye lens. Therefore, the structural study of the dimerization properties of crystallins can shed light on the first stages of protein aggregation. In the present work, we examine the protein-protein association profiles of the human βB2-crystallin by employing extensive coarse-grained molecular dynamics (CG-MD) and the Markov state analysis. Interestingly, our results clearly show important changes in the protein dimerization kinetics between wt-HβB2C and the deamidated systems. The two systems show dimeric conformations. However, the association and dissociation rates are very different. Our results show that the deamidated system can associate faster and dissociate slower than the wt- HβB2C. The deamidated system is in a slightly opened conformation with the Greek-key motifs well folded, suggesting that a complete unfolding of the protein is not required for aggregation. Our results describe the first stages of crystallin aggregation due to post-translational modifications.

The dependency of slow CLC-ec1 dimerization kinetics on lipid solvent volume.

Although protein association equilibrium has been vastly studied in water, questions remain about how membrane proteins assemble and operate within the oily solvent environment of the membrane. However, recent developments in the field provide the potential for understanding the molecular basis of these reactions. In particular, our lab has developed approaches for quantifying the equilibrium affinities of membrane protein oligomers in membranes, a physical model for protein association and folding. Using single-molecule photobleaching microscopy, the lab has demonstrated CLC-ec1 participates in a fully reversible, equilibrium dimerization reaction in membranes. Our studies reveal the kinetics of dimerization are slower than most biological reactions taking days to equilibrate at 37 ºC in 2:1 POPE/POPG lipid bilayers. Here, we investigate the molecular basis for the slow kinetics by measuring subunit exchange kinetics in membranes. We hypothesize the dimerization reaction involves a high-energy transition state that requires the formation of a vacuum cavity in between the two subunits inaccessible to the surrounding solvent. We expect measuring the reaction in mixed lipid bilayers that include smaller lipoidal molecules will lead to a reduced cavity volume along the reaction pathway, decreasing the transition state energy barrier, which will be observed by speeding up of the reaction kinetics. To test this, CLC-ec1-Cy3 and CLC-ec1-Cy5 subunit exchange equilibration rates are measured by bulk Förster Resonance Energy Transfer at 37 ºC in 2:1 POPE/POPG while titrating benzene, n-Octyl-β-D-Glucoside, and 2:1 PE/PG lysolipids. In parallel, coarse-grained molecular dynamics simulations are carried out on the same mixed lipid systems to calculate the solvent-inaccessible cavity volumes between subunits as they are separated from 8 to 24 Å. The results from this investigation will provide a quantitative analysis offering insight into why membrane protein complexes are often kinetically trapped in lipid bilayers.

DNA-induces intra- and inter-molecular changes in dimerization of the human FOXP1.

Transcription factors (TFs) are proteins that regulate gene expression by binding to specific DNA sequences and gating access to genes. This paradigm implies a one-directional gene regulation of DNA remodeling via TFs and their cofactors. Here, we present an example in which the DNA modulates the kinetics of dimerization of the forkhead (FKH) domain of the FoxP1 human TF, favoring its monomeric form over its domain-swapped dimer conformation. Hence, challenging the one-dimensional remodeling paradigm. To inquire about the structural dynamics of the FKH domain in its dimeric form and the presence of DNA, we use molecular dynamics simulations, single-molecule multiparameter fluorescence spectroscopy, and biochemical assays. We found that the monomeric form of the FoxP1 FKH domain primarily displays a disordered-like behavior, which is allosterically reverted by dimerization. Interestingly, DNA favors its interaction with the FKH in its monomeric form over its domain-swapped dimer at the cost of decreasing the FKH domain structural stability. Our findings imply that gene expression is tightly regulated by a negative feedback control over suppressor proteins such as FoxP1 by introducing a bidirectional control over gene expression. Moreover, it suggests that FoxP1 gene suppression action in its domain-swapped conformation, which brings together distant DNA segments and locks transcription access, requires the presence of other dimerization domains, cofactors, or the nuclear macromolecular composition to revert the negative feedback exerted by the DNA.

Study on depolymerization kinetics of formic acid dimers in binary mixture.

In this study, polarization Raman spectra were collected for binary mixtures of formic acid/methanol and formic acid/acetonitrile with different volume fractions. The broad band of formic acid in the CO vibration region was divided into four vibration peaks, corresponding to CO symmetric and anti-symmetric stretching vibration from cyclic dimer, CO stretching from open dimer, and CO stretching from the free monomer. The experiments showed that as the volume fraction of formic acid in the binary mixture decreased, the cyclic dimer gradually converted to the open dimer, and at a volume fraction of 0.1, fully depolymerized into monomer form (free monomer, solvated monomer, and hydrogen bonding monomer clusters with solvent). The contribution percentage of the total CO stretching intensity of each structure at different concentrations was quantitatively calculated using high resolution infrared spectroscopy, and the results were consistent with the conclusions predicted by polarization Raman spectroscopy. Concentration-triggered 2D-COS synchronous and asynchronous spectra also confirmed the kinetics of formic acid diluted in acetonitrile. This work provides a spectroscopic method for studying the structure of organic compounds in solution and concentration-triggering kinetics in mixtures.

On dimerization kinetics and boundary layer transport in crystal growth from dimers

Many molecular crystals are comprised of centrosymmetric dimer motifs. In several cases, these are thought to grow by direct incorporation of dimerized units rather than from monomers. In this work, we model the effects of the dimerization kinetics in the boundary layer close to the crystal surface on the rate of crystal growth. We identify three dimensionless parameters that relate diffusivity through the boundary layer, dimerization kinetics, and dimer incorporation kinetics to estimate the extent to which dimerization kinetics impede the crystal growth from dimers.

Protein Dimerization via Tyr Residues: Highlight of a Slow Process with Co-Existence of Numerous Intermediates and Final Products.

Protein dimerization via tyrosine residues is a crucial process in response to an oxidative attack, which has been identified in many ageing-related pathologies. Recently, it has been found that for isolated tyrosine amino acid, dimerization occurs through three types of tyrosine–tyrosine crosslinks and leads to at least four final products. Herein, considering two protected tyrosine residues, tyrosine-containing peptides and finally proteins, we investigate the dimerization behavior of tyrosine when embedded in a peptidic sequence. After azide radical oxidation and by combining UPLC-MS and H/D exchange analyzes, we were able to evidence: (i) the slow kinetics of Michael Addition Dimers (MAD) formation, i.e., more than 48 h; (ii) the co-existence of intermediates and final cyclized dimer products; and (iii) the probable involvement of amide functions to achieve Michael additions even in proteins. This raises the question of the possible in vivo existence of both intermediates and final entities as well as their toxicity and the potential consequences on protein structure and/or function.

Stimuli-responsive block copolymers as pH chemosensors by fluorescence emission intensification mechanism

Stimuli-responsive block copolymers containing light-responsive coumarin moieties were synthesized using 7-(2-bromoisobutyryloxy)-4-methylcoumarin as an initiator of atom transfer radical polymerization. The resulted smart block copolymers, poly(7-acryloyloxy 4-methylcoumarin-r-methyl methacrylate)-b-poly(dimethylaminoethyl methacrylate) (P(CMA-r-MMA)-b-PDMAEMA) and poly(7-acryloyloxy 4-methylcoumarin)-b-poly(dimethylaminoethyl methacrylate) (PCMA-b-PDMAEMA), were used for pH detection by aggregation-induced fluorescence emission intensification mechanism. Calculation of hydrophilic fraction of the block copolymers by nuclear magnetic resonance spectroscopy proved micellar structure of self-assembled copolymer chains in aqueous media. Dimerization kinetics of the coumarin moieties in the micellar assemblies was investigated by ultraviolet–visible spectroscopy at different pH values. The resulted aqueous micellar dispersions of the block copolymers with different compositions were utilized for pH detection by using fluorescence spectroscopy. The copolymer containing MMA units as a spacer between coumarin moieties (P(CMA-r-MMA)-b-PDMAEMA) showed higher dimerization kinetics and applicability as a fluorescence chemosensor for detection of pH. By contraction of PDMAEMA blocks at higher pH values and consequent aggregation state of the coumarin moieties in the core, the change of fluorescence intensity was used for fluorescence sensation of pH.

Time-resolved detection of association/dissociation reactions and conformation changes in photosensor proteins for application in optogenetics.

Photosensor proteins are important not only because of their biological functions but also because of their applications in optogenetics. To understand the molecular mechanism behind their biological functions and consequently seek possible applications to optogenetics, the dynamics of their intermolecular interaction (for example, association/dissociation reaction and conformational changes) upon photoexcitation need to be elucidated. Although it has been difficult to trace such reactions in the time domain using traditional spectroscopic techniques, the time-resolved diffusion method based on the transient grating technique has been demonstrated to possess a significant advantage in detecting such spectrally silent dynamics in a time-resolved manner. In this paper, the principle and studies on blue light sensor proteins, phototropins, are described. Reaction kinetics of dimerization, dissociation reactions, and conformational changes were measured, and reaction schemes were determined. This method can be employed to study protein reactions from the viewpoint of diffusion and to elucidate the reaction schemes and kinetics that cannot be detected by other spectroscopic methods.

Smart block copolymers as fluorescence chemosensors of copper ions with high detection limit

Stimuli-responsive block copolymers were synthesized to detect copper ions with high limit based on a fluorescence quenching mechanism. For this purpose, poly(7-acryloyloxy 4-methylcoumarin-r-methyl methacrylate)-b-poly(dimethylaminoethyl methacrylate) in three different block ratios was synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. Successful synthesis of the block copolymers was confirmed by Fourier-transformed infrared and proton nuclear magnetic resonance spectroscopies. Self-assembly of the copolymers in aqueous media resulted in formation of micellar structures, as confirmed by transmission electron microscopy images and also estimated form the hydrophilic fraction of the block copolymers. Micellar size and dispersity were studied by dynamic light scattering in different conditions. In addition, dimerization kinetics of the coumarin molecules in the micellar assemblies were studied by ultraviolet–visible spectroscopy. The sample with higher molar ratio of CMA in the hydrophobic block and shorter length of PDMAEMA in the hydrophilic block showed faster dimerization reaction. The micellar assemblies formed from all the copolymers were used to sense potassium, calcium, and copper cations. All the self-assembled structures showed high sensitivity to detect copper cation, by quenching their fluorescence intensity. The copolymer sample with the highest ratio of PDMAEMA/CMA in the chain structure has the highest detection limit of 0.002 ppm for copper ion.

Human FoxP Transcription Factors as Tractable Models of the Evolution and Functional Outcomes of Three-Dimensional Domain Swapping.

The association of two or more proteins to adopt a quaternary complex is one of the most widespread mechanisms by which protein function is modulated. In this scenario, three-dimensional domain swapping (3D-DS) constitutes one plausible pathway for the evolution of protein oligomerization that exploits readily available intramolecular contacts to be established in an intermolecular fashion. However, analysis of the oligomerization kinetics and thermodynamics of most extant 3D-DS proteins shows its dependence on protein unfolding, obscuring the elucidation of the emergence of 3D-DS during evolution, its occurrence under physiological conditions, and its biological relevance. Here, we describe the human FoxP subfamily of transcription factors as a feasible model to study the evolution of 3D-DS, due to their significantly faster dissociation and dimerization kinetics and lower dissociation constants in comparison to most 3D-DS models. Through the biophysical and functional characterization of FoxP proteins, relevant structural aspects highlighting the evolutionary adaptations of these proteins to enable efficient 3D-DS have been ascertained. Most biophysical studies on FoxP suggest that the dynamics of the polypeptide chain are crucial to decrease the energy barrier of 3D-DS, enabling its fast oligomerization under physiological conditions. Moreover, comparison of biophysical parameters between human FoxP proteins in the context of their minute sequence differences suggests differential evolutionary strategies to favor homoassociation and presages the possibility of heteroassociations, with direct impacts in their gene regulation function.

Cosolute modulation of protein oligomerization reactions in the homeostatic timescale

Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24–48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction.

Cu(I) Reducibility Controls Ethylene vs Ethanol Selectivity on (100)-Textured Copper during Pulsed CO2 Reduction.

The electrochemical CO2 reduction reaction (CO2RR) can convert widely available CO2 into value-added C2 products, such as ethylene and ethanol. However, low selectivity toward either compound limits the effectiveness of current CO2RR electrocatalysts. Here, we report the use of pulsed overpotentials to improve the ethylene selectivity to 67% with >75% overall C2 selectivity on (100)-textured polycrystalline Cu foil. The pulsed CO2RR can be made selective to either ethylene or ethanol by controlling the reaction temperature. We attribute the enhanced C2 selectivity to the improved CO dimerization kinetics on the active Cu surface on predominately (100)-textured Cu grains with the reduced hydrogen adsorption coverage during the pulsed CO2RR. The ethylene vs ethanol selectivity can be explained by the reducibility of the Cu(I) species during the cathodic potential cycle. Our work demonstrates a simple route to improve the ethylene vs ethanol selectivity and identifies Cu(I) as the species responsible for ethanol production.

Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer.

The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA-p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA-RelA homodimer is critical as it facilitates the formation of the more stable RelA-p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA-RelA homodimer, the kinetics of the RelA-p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA-RelA specific target gene regulation in physiology.

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

A Plasmonic Approach to Study Protein Interaction Kinetics through the Dimerization of Functionalized Ag Nanoparticles

Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag NPs) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. To illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-STV) pair interaction was studied. Controlled aggregates (dimers) of STV functionalized Ag NPs were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag NPs, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.

Effect of protein cryoconcentration and processing conditions on kinetics of dimer formation for a monoclonal antibody: A case study on bioprocessing.

Monoclonal antibodies (mAbs) may be prone to self-association leading to formation of dimers, trimers, or other high molecular weight species during bio-processing. In order to implement appropriate manufacturing control strategies during bio-processing, it is important to understand various real life bio-processing conditions where such self-associations may manifest. One such case study is presented here of increase in dimer content for an mAb during scale-up bio-processing and the approach taken to understand the under-lying mechanism. In this example, a therapeutic mAb demonstrated a consistently higher dimer values (~0.5% higher) in the drug product (DP) during release when compared to the same value measured in the corresponding drug substance (DS) lot. This observation was interesting since the DS was supplied frozen, and the DS and DP share the same formulation composition and therefore investigation of this dimer change was the scope of the characterization study. Variable path length spectroscopy and size exclusion chromatography was used for protein quantification and to monitor %dimer respectively during characterization of fill-finish unit operations. At the start of DP manufacturing process, immediately after thaw of bulk DS, a protein concentration gradient was observed and the concentration ranged from 90 mg/mL (top of container) to 210 mg/mL (bottom of container). The dimerization kinetics in the same DS container was dependent on concentration with higher concentrations demonstrating higher rates of dimerization. After the bulk DS was mixed for further processing, %dimer in purified bulk DS was quantitated to be approximately 1.4% which is identical to levels observed during scale-up manufacturing of DP. After each unit operation, the in-process samples tested for %dimer showed a gradual increase in dimer as a function of time over the next 7 days accumulating to 1.8% dimer at the end of DP manufacturing process. Samples subjected to static incubation at 2-8°C and room temperature (RT; 15-25°C) showed a gradual increase in dimer over the same time frame; however, the rate of increase in dimer at RT was higher compared to samples stored at 2-8°C. The results from this demonstrate two important key findings: self-association kinetics of mAbs could be exacerbated by protein cryoconcentration and temperature conditions during bioprocessing. Since these two parameters are commonly encountered during manufacturing, the proposed mitigation strategy is to ensure homogeneity of the bulk DP during processing. The temperature dependent self-association kinetics of mAb could be mitigated by processing at lower temperature (e.g., 2-8°C) and by storing the finished DP at lower temperature after manufacturing. The results from this study also highlight the criticality of setting slightly wider specifications for DP compared to DS following ICH Q6B guidelines.