Oligomerization Behavior Research Articles

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm2/s), slow (∼0.04 μm2/s), and fast (∼0.14 μm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations

DNA methyltransferases DNMT3A- and DNMT3B-mediated DNA methylation critically regulate epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote polymerization, leading to aberrant DNA methylation that may contribute to the pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the mutation-induced functional misregulation of DNMT3A remains unclear. Here, we report the crystal structures of the DNMT3A methyltransferase domain, revealing a molecular basis for its oligomerization behavior distinct to DNMT3B, and the enhanced intermolecular contacts caused by the R882H or R882C mutation. Our biochemical, cellular, and genomic DNA methylation analyses demonstrate that introducing the DNMT3B-converting mutations inhibits the R882H-/R882C-triggered DNMT3A polymerization and enhances substrate access, thereby eliminating the dominant-negative effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutations-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.

Biochemical, structural, and computational analyses of two new clinically identified missense mutations of ALDH7A1

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes a step of lysine catabolism. Certain missense mutations in the ALDH7A1 gene cause pyridoxine dependent epilepsy (PDE), a rare autosomal neurometabolic disorder with recessive inheritance that affects almost 1:65,000 live births and is classically characterized by recurrent seizures from the neonatal period. We report a biochemical, structural, and computational study of two novel ALDH7A1 missense mutations that were identified in a child with rare recurrent seizures from the third month of life. The mutations affect two residues in the oligomer interfaces of ALDH7A1, Arg134 and Arg441 (Arg162 and Arg469 in the HGVS nomenclature). The corresponding enzyme variants R134S and R441C (p.Arg162Ser and p.Arg469Cys in the HGVS nomenclature) were expressed in Escherichia coli and purified. R134S and R441C have 10,000- and 50-fold lower catalytic efficiency than wild-type ALDH7A1, respectively. Sedimentation velocity analytical ultracentrifugation shows that R134S is defective in tetramerization, remaining locked in a dimeric state even in the presence of the tetramer-inducing coenzyme NAD+. Because the tetramer is the active form of ALDH7A1, the defect in oligomerization explains the very low catalytic activity of R134S. In contrast, R441C exhibits wild-type oligomerization behavior, and the 2.0 Å resolution crystal structure of R441C complexed with NAD+ revealed no obvious structural perturbations when compared to the wild-type enzyme structure. Molecular dynamics simulations suggest that the mutation of Arg441 to Cys may increase intersubunit ion pairs and alter the dynamics of the active site gate. Our biochemical, structural, and computational data on two novel clinical variants of ALDH7A1 add to the complexity of the molecular determinants underlying pyridoxine dependent epilepsy.

PH Dependence of HSF1 trimerization is shaped by intramolecular interactions

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Zipper-Confined DNA Nanoframe for High-Efficient and High-Contrast Imaging of Heterogeneous Tumor Cell.

Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics.

Charge manipulation of the human insulin B chain C-terminal to shed light on the complex mechanism of insulin fibrillation

Insulin fibrillation poses a significant challenge in the development and treatment of diabetes. Current efforts to unravel its mechanisms have thus far remained incomplete. To shed light on the intricate processes behind insulin fibrillation, we employed mutagenesis techniques to introduce additional positive charge residues into the C-terminal region of the insulin B chain which plays an important role in insulin dimerization. We employed our investigation with various spectroscopic methods, electron microscopy, and molecular dynamics simulations. These methods allowed us to explore the structure and fibrillation behavior of the engineered B chains following their expression in a bacterial host and successful purification. This manipulation had a pronounced impact on the oligomerization behavior of the insulin B chain. It appears that these mutations delay the formation of the dimeric state in the process of transitioning to larger oligomers, consequently, leading to an alteration in the kinetics of fibrillation. Our findings also indicated that the mutant insulin B chains (Di-R, Di-K, and Di-H) displayed resistance to the initiation of fibrillation. This resistance can be attributed to the repulsive forces generated by the introduced positive charges, which disrupt the attractive interactions favoring nucleation. Notably, the mutant B chains formed shorter and less abundant oligomers and fibrils, which can be ascribed to the alterations induced by repulsion. Our engineered mutant B chains exhibited enhanced stability against stress-induced fibrillation, hinting at their potential utility in the development of new insulin analogs. This study underscores the significance of the C-terminal region in the initial stages of insulin B chain fibrillation, providing valuable insights into the intricate mechanisms involved and their potential pharmaceutical applications.

Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs.

Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

Interspecies Variation Affects Islet Amyloid Polypeptide Membrane Binding.

The aggregation of islet amyloid polypeptide (IAPP) is associated with β-cell dysfunction in type 2 diabetes (T2D) in humans. One possible mechanism of toxicity is the interaction of IAPP oligomers with lipid membranes to disrupt the bilayer integrity and/or homeostasis of the cell. Amino acid sequence variations of IAPPs between species can greatly decrease their propensity for aggregation. For example, human IAPP is toxic to β-cells, but rat and pig IAPP are not. However, it is not clear how these differences affect membrane association. Using native mass spectrometry with lipid nanodiscs, we explored the differences in the association of human, rat, and pig IAPP with lipid bilayers. We discovered that human and rat IAPP bound nanodiscs with anionic dipalmitoyl-phosphatidylglycerol (DPPG) lipids, but pig IAPP did not. Furthermore, human and rat IAPP interacted differently with the membrane. Human IAPP show potential tetramer complexes, but rat IAPP associated with the membrane sequentially. Thus, overall IAPP-bilayer interactions are not necessarily related to disease, but small differences in oligomeric behavior at the membrane may instead play a role.

Molecular characterization of phototropin signalosome regulating photophysiological processes in green algae.

Phototropin is a blue light-sensing photoreceptor, comprises two LOV domains that non-covalently bind FMN as chromophore and a serine-threonine kinase domain at its C-terminus. Phototropin is well established in higher plants and aquatic algae. However, the photoreceptor biology of the evolutionary important alga has not yet been established. We characterized the phototropin LOV domain from evolutionary important algae and compared the changes with the aquatic green algae, Chlamydomonas reinhardtii phototropin LOV domain. The intraflagellar transport (IFT) machinery responsible for bidirectional movement of proteins inwithin cilia or flagella is involved in trafficking of Chlamydomonas phototropin (CrPhot). Through immunoblotting and immunolocalization experiments with a variety of IFT mutants, we investigated the molecular details of phototropin trafficking. The CrPhot knock-out strains showed reduced photomotility, decrease level of other predicted interacting proteins like ChRs and, 14-3-3 as compared to that of the wild type strain. Various approaches like protein profiling, expression profile (qRT-PCR), and proteomics were applied to delineate its role in light-mediated physiology in C. reinhardtii. We studied the photochemistry, oligomerization, and photodynamic behaviour of LOV1 domain of a novel terrestrial algal phototropin using UV-visible, fluorescence, CD, atomic force microscopy, and time-resolved fluorescence spectroscopy techniques. Our findings highlight the unique characteristics of LOV domain in terms of very long dark recovery time. We identified the key residues responsible for this recovery kinetics in novel LOV1 protein. Different variants of LOV1 protein were produced and photochemistry was compared with the respect to the wild type protein. We proposed optogenetic potential of the engineered LOV domain in modulation of diverse cellular signalling pathways such as ciliogenesis, cell cycle, and apoptosis.

α-Crystallin Domains of Five Human Small Heat Shock Proteins (sHsps) Differ in Dimer Stabilities and Ability to Incorporate Themselves into Oligomers of Full-Length sHsps

The α-crystallin domain (ACD) is the hallmark of a diverse family of small heat shock proteins (sHsps). We investigated some of the ACD properties of five human sHsps as well as their interactions with different full-length sHsps. According to size-exclusion chromatography, at high concentrations, the ACDs of HspB1 (B1ACD), HspB5 (B5ACD) and HspB6 (B6ACD) formed dimers of different stabilities, which, upon dilution, dissociated to monomers to different degrees. Upon dilution, the B1ACD dimers possessed the highest stabilities, and those of B6ACD had the lowest. In striking contrast, the ACDs of HspB7 (B7ACD) and HspB8 (B8ACD) formed monomers in the same concentration range, which indicated the compromised stabilities of their dimer interfaces. B1ACD, B5ACD and B6ACD transiently interacted with full-length HspB1 and HspB5, which are known to form large oligomers, and modulated their oligomerization behavior. The small oligomers formed by the 3D mutant of HspB1 (mimicking phosphorylation at Ser15, Ser78 and Ser82) effectively interacted with B1ACD, B5ACD and B6ACD, incorporating these α-crystallin domains into their structures. The inherently dimeric full-length HspB6 readily formed heterooligomeric complexes with B1ACD and B5ACD. In sharp contrast to the abovementioned ACDs, B7ACD and B8ACD were unable to interact with full-length HspB1, the 3D mutant of HspB1, HspB5 or HspB6. Thus, their high sequence homology notwithstanding, B7ACD and B8ACD differ from the other three ACDs in their inability to form dimers and interact with the full-length small heat shock proteins. Having conservative primary structures and being apparently similar, the ACDs of the different sHsps differ in terms of their dimer stabilities, which can influence the heterooligomerization preferences of sHsps.

Influence of Bridged Group on Coordination Mode and Ethylene Oligomerization Behavior of Diamine Bridged Salicylaldimine Ni(II) Complexes

Influence of Bridged Group on Coordination Mode and Ethylene Oligomerization Behavior of Diamine Bridged Salicylaldimine Ni(II) Complexes

Chromium-Based Complexes Bearing N-Substituted Diphosphinoamine Ligands for Ethylene Oligomerization.

A range of novel N-substituted diphosphinoamine (PNP) ligands Ph2PN(R)PPh2 [R = F2CHCH2 (1); R = Me2CHCH2 (2); R = Me2CHCH2CH2 (3)] have been synthesized via one-step salt elimination reaction. The ligand-coordinated chromium carbonyls [Ph2PN(R)PPh2]Cr(CO)4 (4–6) were further synthesized, and X-ray crystallography analysis of complex 6 revealed the κ2-P,P bidentate binding mode of Cr center and the molecular structure of PNP ligand 3. Then the catalytic ethylene oligomerization behaviors of PNP ligands 1–3 bridging chromium chloride complexes {[Ph2PN(R)PPh2]CrCl2(μ-Cl)}2 (7–9) were further discussed in depth. Experimental results showed that complex 7 with the strong electron-withdrawing F2CHCH2 group can promote the nonselective ethylene oligomerization, while both complex 8 and complex 9 with the electron-donating Me2CHCH2 and Me2CHCH2CH2 groups can significantly enhance the selective ethylene tri/tetramerization. The good catalytic activity of 198.3 kg/(g Cr·h), the selectivity toward 1-hexene and 1-octene of 76.4%, and the low PE content of 0.2% were simultaneously achieved with the Al/Cr molar ratio of 600 using the complex 8/MMAO system at 45 °C and 45 bar. These excellent results were mainly attributed to the fact that the β-branching of bridging ligand 2 increased the steric bulk of the N-moiety for complex 8.

Single Molecule Analysis Reveals Dimer Formation of the Norepinephrine Transporter at the Plasma Membrane of Live Cells

BackgroundMonoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug‐induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co‐exists as monomers, dimers, and higher‐order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)‐bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.MethodsWe fused monomeric green fluorescent protein (mGFP) to the N‐terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.ResultsSingle molecule brightness analysis revealed that NET co‐exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Student's t‐test, P<0.05).ConclusionNET forms monomers and dimers but no higher‐order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.

Synthesis and ethylene oligomerization behavior of trinuclear nickel complex with phosphorus dendrimer

Synthesis and ethylene oligomerization behavior of trinuclear nickel complex with phosphorus dendrimer

Structural Studies Providing Insights into Production and Conformational Behavior of Amyloid-β Peptide Associated with Alzheimer's Disease Development.

Alzheimer’s disease is the most common type of neurodegenerative disease in the world. Genetic evidence strongly suggests that aberrant generation, aggregation, and/or clearance of neurotoxic amyloid-β peptides (Aβ) triggers the disease. Aβ accumulates at the points of contact of neurons in ordered cords and fibrils, forming the so-called senile plaques. Aβ isoforms of different lengths are found in healthy human brains regardless of age and appear to play a role in signaling pathways in the brain and to have neuroprotective properties at low concentrations. In recent years, different substances have been developed targeting Aβ production, aggregation, interaction with other molecules, and clearance, including peptide-based drugs. Aβ is a product of sequential cleavage of the membrane glycoprotein APP (amyloid precursor protein) by β- and γ-secretases. A number of familial mutations causing an early onset of the disease have been identified in the APP, especially in its transmembrane domain. The mutations are reported to influence the production, oligomerization, and conformational behavior of Aβ peptides. This review highlights the results of structural studies of the main proteins involved in Alzheimer’s disease pathogenesis and the molecular mechanisms by which perspective therapeutic substances can affect Aβ production and nucleation.

In-solution structural studies involving a phospholipase A2-like myotoxin and a natural inhibitor: Plasticity of oligomeric assembly affects mechanisms of inhibition

Snakebite envenomation has been categorized by World Health Organization as a category A neglected tropical disease, since it causes chronic psychological disorders, physical disablement and death. Ophidian accidents may cause local myonecrosis that cause drastic sequelae, which are not efficiently neutralized via serum therapy. Phospholipase A2-like (PLA2-like) myotoxins have a major role in the local effects caused by several snake venoms. We previously demonstrated that chicoric acid (CA) is an efficient inhibitor of the BthTX-I myotoxin and solved the X-ray structure of complex. Herein, we assess the oligomeric behavior of the BthTX-I/CA complex in solution under different physical-chemical conditions and using toxin obtained by two different biochemical methodologies to fully elucidate structural bases of inhibition of myotoxins by CA. We demonstrated the ability of PLA2-like proteins to form different oligomeric assemblies in the presence of certain inhibitors, which can also be modulated by buffer polarity change. In the presence of ethanol, BthTX-I/CA remains predominantly in a monomeric conformation, which prevents it from being in its active form (dimeric conformation). In contrast, in the absence of ethanol, the tetramer assembly was observed, which hid key regions of the protein responsible for docking and disruption of the muscle membrane. Therefore, the “plasticity” of these proteins with regard to their abilities to form oligomeric assemblies is a key issue for the future development of therapeutic agents to complement of serum therapy.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68–ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to ‘catch’ the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBMCipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of 90 ± 30 μM. As the KD of CBMCipA binding to cellulose is below 1 μM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBMCipA, its dimerization is likely to have a marked effect on its solution behavior.

Evidence for different in vitro oligomerization behaviors of synthetic hIAPP obtained from different sources.

Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process. Graphical abstract.