Formation Of Protein Oligomers Research Articles

Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

Unveiling the structural and functional consequences of the p.D109G pathogenic mutation in human αB-Crystallin responsible for restrictive cardiomyopathy and skeletal myopathy

αB-Crystallin (αB-Cry) is expressed in many tissues, and mutations in this protein are linked to various diseases, including cataracts, Alzheimer's disease, Parkinson's disease, and several types of myopathies and cardiomyopathies. The p.D109G mutation, which substitutes a conserved aspartate residue involved in the interchain salt bridges, with glycine leads to the development of both restrictive cardiomyopathy (RCM) and skeletal myopathy. In this study, we generated this mutation in the α-Cry domain (ACD) which is crucial for forming the active chaperone dimeric state, using site-directed mutagenesis. After inducing expression in the bacterial host, we purified the mutant and wild-type recombinant proteins using anion exchange chromatography. Various spectroscopic evaluations revealed significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry caused by this mutation. Furthermore, this pathogenic mutation led to the formation of protein oligomers with larger sizes than those of the wild-type protein counterpart. The mutant protein also exhibited increased chaperone activity and decreased chemical, thermal, and proteolytic stability. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence microscopy (FM) demonstrated that p.D109G mutant protein is more prone to forming amyloid aggregates. The misfolding associated with the p.D109G mutation may result in abnormal interactions of human αB-Cry with its natural partners (e.g., desmin), leading to the formation of protein aggregates. These aggregates can interfere with normal cellular processes and may contribute to muscle cell dysfunction and damage, resulting in the pathogenic involvement of the p.D109G mutant protein in restrictive cardiomyopathy and skeletal myopathy.

Radial pair correlation of molecular brightness fluctuations maps protein diffusion as a function of oligomeric state within live-cell nuclear architecture

Nuclear proteins can modulate their DNA binding activity and the exploration volume available during DNA target search by self-associating into higher-order oligomers. Directly tracking this process in the nucleoplasm of a living cell is, however, a complex task. Thus, here we present a microscopy method based on radial pair correlation of molecular brightness fluctuations (radial pCOMB) that can extract the mobility of a fluorescently tagged nuclear protein as a function of its oligomeric state and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture. By simply performing a rapid frame scan acquisition, radial pCOMB has the capacity to detect, within each pixel, protein oligomer formation and the size-dependent obstruction nuclear architecture imparts on this complex’s transport across sub-micrometer distances. From application of radial pCOMB to an oligomeric transcription factor and DNA repair protein, we demonstrate that homo-oligomer formation differentially regulates chromatin accessibility and interaction with the DNA template.

Use of 3D domain swapping in constructing supramolecular metalloproteins.

Supramolecules, which are formed by assembling multiple molecules by noncovalent intermolecular interactions instead of covalent bonds, often show additional properties that cannot be exhibited by a single molecule. Supramolecules have evolved into molecular machines in the field of chemistry, and various supramolecular proteins are responsible for life activities in the field of biology. The design and creation of supramolecular proteins will lead to development of new enzymes, functional biomaterials, drug delivery systems, etc.; thus, the number of studies on the regulation of supramolecular proteins is increasing year by year. Several methods, including disulfide bond, metal coordination, and surface-surface interaction, have been utilized to construct supramolecular proteins. In nature, proteins have been shown to form oligomers by 3D domain swapping (3D-DS), a phenomenon in which a structural region is exchanged between molecules of the same protein. We have been studying the mechanism of 3D-DS and utilizing 3D-DS to construct supramolecular metalloproteins. Cytochrome c forms cyclic oligomers and polymers by 3D-DS, whereas other metalloproteins, such as various c-type cytochromes and azurin form small oligomers and myoglobin forms a compact dimer. We have also utilized 3D-DS to construct heterodimers with different active sites, a protein nanocage encapsulating a Zn-SO4 cluster in the internal cavity, and a tetrahedron with a designed building block protein. Protein oligomer formation was controlled for the 3D-DS dimer of a dimer-monomer transition protein. This article reviews our research on supramolecular metalloproteins.

P.473 BDNF and NGF as a key neuroplasticity factors in patients with ischemic stroke

Enzymes and/or proteins are commonly present in oligomeric forms for active biological functions. The formation of protein oligomers can take place via covalent bonding or through weak-bond-network associations. Oligomerization can occur when stable proteins unfold, creating exposed interfaces for association interactions. One well-known process of weak-bond-network oligomerization is domain swapping (DS). The occurrence of oligomerization is usually in response to environmental changes such as PH and temperature. Oligomeric enzymes provide homosteric active centers (or binding sites). Interactive enzymes can also be allosteric, in that multiple active centers have distinctly different ligand binding affinities. In general, ligand binding on an interactive protein/enzyme results in the protein/enzyme to undergo conformal structural changes. When an enzyme molecule is free of ligands, the enzyme rests in a particular stable form. If one ligand molecule is bound on the enzyme molecule, the enzyme-ligand complex can exhibit another conformal structure that is different from that of the ligand-free enzyme. As more ligands are added to the enzyme-ligand complex, the enzyme with ligands bound can exhibit successively different conformal structures. Therefore, even a homosteric interactive enzyme can exhibit variable affinity among different active centers. Intricate and complex molecular interactions form the bases for complex biological functions. Treaties have been developed to quantitatively describe the behaviors of interactive enzymes with single and multiple ligands.

Chitooligosaccharide binding to CIA17 (Coccinia indica agglutinin). Thermodynamic characterization and formation of higher order complexes

CIA17 is a PP2-like, homodimeric lectin made up of 17 kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)2–6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (Ka = 1.8 × 107 M−1) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)6-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.

Direct observation of prion protein oligomer formation reveals an aggregation mechanism with multiple conformationally distinct species.

The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases. In the past decade, a similar process has been associated with other proteins, such as Aβ, tau, and α-synuclein, which participate in other neurodegenerative diseases. It is increasingly recognized that the small oligomeric species of aggregates can play an important role in the development of prion diseases. However, determining the nature of the oligomers formed during the aggregation process has been experimentally difficult due to the lack of suitable methods capable of the detection and characterization of the low level of oligomers that may form. To address this problem, we have utilized single-aggregate methods to study the early events associated with aggregation of recombinant murine PrP in vitro to approach the bona fide process in vivo. PrP aggregation resulted in the formation of thioflavin T (ThT)-inactive and ThT-active species of oligomers. The ThT-active oligomers undergo conversion from a Proteinase K (PK)-sensitive to PK-resistant conformer, from which mature fibrils can eventually emerge. Overall, our results show that single-aggregate methods can provide structural and mechanistic insights into PrP aggregation, identify the potential species that mediates cytotoxicity, and reveal that a range of distinct oligomeric species with different properties is formed during prion protein aggregation.

SAR by kinetics for drug discovery in protein misfolding diseases

To develop effective therapeutic strategies for protein misfolding diseases, a promising route is to identify compounds that inhibit the formation of protein oligomers. To achieve this goal, we report a structure-activity relationship (SAR) approach based on chemical kinetics to estimate quantitatively how small molecules modify the reactive flux toward oligomers. We use this estimate to derive chemical rules in the case of the amyloid beta peptide (Aβ), which we then exploit to optimize starting compounds to curtail Aβ oligomer formation. We demonstrate this approach by converting an inactive rhodanine compound into an effective inhibitor of Aβ oligomer formation by generating chemical derivatives in a systematic manner. These results provide an initial demonstration of the potential of drug discovery strategies based on targeting directly the production of protein oligomers.

Formation and carbon monoxide-dependent dissociation of Allochromatium vinosum cytochrome c' oligomers using domain-swapped dimers.

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c' from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.

Gas-phase protein conformation/multimer ion formation by electrospray ion mobility-mass spectrometry: bovine insulin and ubiquitin.

Ion mobility-mass spectrometry (IMMS) is a very attractive method for studies in structural biology because of the ability of rapid isolation by nearly simultaneous m/z characterization and size separation, leading to an emergence of IMMS as a complimentary biochemical tool. Earlier, we developed a method based on varying the protein concentration in solution prior to electrospray ionization (ESI) with subsequent m/z selection and dissociation of protein multimers by IMMS of cytochrome c. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z for well-studied proteins bovine ubiquitin and insulin. These proteins were chosen due to their large difference in solution phase structures: insulin tightly bound by disulfide linkages, and ubiquitin-a protein that may adopt a range of states from compact to extended. Our preliminary results, as with cytochrome c reveal false negatives for protein oligomer formation and false positives for protein conformational states. In addition, these results will be couched in terms of the need for quantification of IMMS analysis of proteins given the total area under IMMS peaks can also distinguish conformation versus aggregation as higher order oligomers have more mass per ion.This article is part of the themed issue 'Quantitative mass spectrometry'.

A Multi-Scale Model for the Assembling Kinetics of Protein Complexes

The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies show numerous examples that misassembly of protein complexes can lead to severe biological consequences. Although our research focus has started to be moved beyond the static representation to the dynamic aspect of protein quaternary structures, current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multi-scale modeling framework. The framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method, so that protein complex can be assembled with both structural details and computational efficiency. We applied the model to a homo-trimer and a hetero-tetramer as simple testing systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multi-step process that is much slower than dimerization, but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunit, which is a significant feature to regulate functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without taking accounts of allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly.

Multiscale Model for the Assembly Kinetics of Protein Complexes.

The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly.

A review on protein oligomerization process

Enzymes or proteins are commonly present in oligomeric forms for active biological functions. The formation of protein oligomers, either in nature or by artificial means, can take place via covalent bonding or through weak-bond-network associations. Disulfide bonds are more common in forming covalent protein oligomers. Covalent bound protein oligomers usually have additional elements introduced, while proteins associated through weak-bond-networks usually do not involve any additional bound chemical groups. Proteins are commonly present in stable forms or folds. Oligomerization can occur when stable proteins unfold, creating exposed interfaces for association interactions. One well-known process of weak-bond-network oligomerization is domain swapping (DS). Separating the two touching/interacting domains creates opportunities for similar interactions with different protein molecules, leading to oligomerization. Both disulfide bonding and DS oligomerization can be dynamic (or reversible) at least at low Degree of Polymerizations (DPs). The dynamic nature of the protein oligomerization is important for bioactivity control. The morpheein model and the polymorph model are thus important in quantifying enzyme catalyzed biotransformations. Disulfide bonding and DS can act together to form supramolecules. The formation of supramolecules, such as amyloids, in nature can be benign or harmful. Industrial applications of protein oligomerization exploit the special functions /properties of the resultant supramolecules, such as multiple biofunctions, niche structure, etc..

A Role for Autoinhibition in Preventing Dimerization of the Transcription Factor ETS1

ETS1 is the archetype of the ETS transcription factor (TF) family. ETS TFs share a DNA-binding domain, the ETS domain. All ETS TFs recognize a core GGA(A/T) binding site, and thus ETS TFs are found to redundantly regulate the same genes. However, each ETS TF has unique targets as well. One prevailing hypotheses explaining this duality is that protein-protein interactions, including homodimerization, allow each ETS TF to display distinct behavior. The behavior of ETS1 is further regulated by autoinhibition. Autoinhibition apparently modulates ETS1 DNA binding affinity, but the mechanism of this inhibition is not completely understood. We sought to characterize the relationship between DNA binding and ETS1 homodimer formation. We find that ETS1 interrogates DNA and forms dimers even when the DNA does not contain an ETS recognition sequence. Mutational studies also link nonspecific DNA backbone contacts with dimer formation, in addition to providing a new role for the recognition helix of ETS1 in maintaining the autoinhibited state. Finally, in showing that residues in the DNA recognition helix affect autoinhibition, we define a new function of ETS1 autoinhibition: maintenance of a monomeric state in the absence of DNA. The conservation of relevant amino acid residues across all ETS TFs indicates that the mechanisms of nonspecific DNA interrogation and protein oligomer formation elucidated here may be common to all ETS proteins that autoinhibit.

Protein Cross-Linking and Oligomerization through Dityrosine Formation upon Exposure to Ozone.

Air pollution is a potential driver for the increasing prevalence of allergic disease, and post-translational modification by air pollutants can enhance the allergenic potential of proteins. Here, the kinetics and mechanism of protein oligomerization upon ozone (O3) exposure were studied in coated-wall flow tube experiments at environmentally relevant O3 concentrations, relative humidities and protein phase states (amorphous solid, semisolid, and liquid). We observed the formation of protein dimers, trimers, and higher oligomers, and attribute the cross-linking to the formation of covalent intermolecular dityrosine species. The oligomerization proceeds fast on the surface of protein films. In the bulk material, reaction rates are limited by diffusion depending on phase state and humidity. From the experimental data, we derive a chemical mechanism and rate equations for a kinetic multilayer model of surface and bulk reaction enabling the prediction of oligomer formation. Increasing levels of tropospheric O3 in the Anthropocene may promote the formation of protein oligomers with enhanced allergenicity and may thus contribute to the increasing prevalence of allergies.

Co‐existence of Two Different α‐Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry

AbstractNeurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson’s disease involves the protein α‐synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild‐type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co‐existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39–A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.

Co-existence of two different α-synuclein oligomers with different core structures determined by hydrogen/deuterium exchange mass spectrometry.

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson's disease involves the protein α-synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild-type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co-existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D2 O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39-A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.

Regulated Oligomerization Induces Uptake of a Membrane Protein into COPII Vesicles Independent of Its Cytosolic Tail

Export of transmembrane proteins from the endoplasmic reticulum (ER) is driven by directed incorporation into coat protein complex II (COPII)-coated vesicles. The sorting of some cargo proteins into COPII vesicles was shown to be mediated by specific interactions between transmembrane and COPII-coat-forming proteins. But even though some signals for ER exit have been identified on the cytosolic domains of membrane proteins, the general signaling and sorting mechanisms of ER export are still poorly understood. To investigate the role of cargo protein oligomer formation in the export process, we have created a transmembrane fusion protein that-owing to its FK506-binding protein domains-can be oligomerized in isolated membranes by addition of a small-molecule dimerizer. Packaging of the fusion protein into COPII vesicles is strongly enhanced in the presence of the dimerizer, demonstrating that the oligomeric state is an ER export signal for this membrane protein. Surprisingly, the cytosolic tail is not required for this oligomerization-dependent effect on protein sorting. Thus, an alternative mechanism, such as membrane bending, must account for ER export of the fusion protein.

Critical Examination of Gas-Phase Protein Conformation/Multimer Ion Formation by Electrospray Ion Mobility-Mass Spectrometry

The generally accepted view of protein structure in the gas-phase is that protein ions produced by electrospray ionization (ESI) exist in a number of different states, and the resulting charge state distribution (CSD) and ion mobility spectrum is interpreted as evidence for protein ions retaining some memory of solution-phase conformation. Even with the inclusion of ion mobility information, reports of protein ion structure in the gas-phase are oftentimes in disagreement not only within the discipline but also as interpreted by other gas-phase techniques. The focus of this work will be to correctly distinguish truly different ion conformations formed by ESI versus homomultimeric complexes with the same m/z. The concentration of cytochrome c in solution was varied over a wide range, and the multiply charged multimers (MCMs) present in the ion mobility/mass spectrum were unambiguously assigned by m/z selection and dissociation prior to ion mobility/mass spectrometry analysis. The results revealed false negatives for protein oligomer formation and false positives for protein conformational states and no evidence that gas-phase cytochrome c ions retain memory of solution-phase conformation, characteristics of great importance for structural biology. The results also suggest that the total IM-MS distribution for a protein is the complex result of individual MCMs either surviving until detection (undissociated) or dissociating into lower order multimers or a number of product ions for each m/z.