Oligomeric Structure Research Articles

The Multifaceted Subunit Interface of Malate Dehydrogenase

Most Malate Dehydrogenases are dimeric though there are a small number (for example the LDH like Apicomplexan Malate Dehydrogenases) with tetrameric structures. A variety of studies have shown the oligomeric structure to be important for the overall activity of the enzyme with roles from stability to allosteric interactions being suggested. To explore our overall hypothesis that regions of the subunit interface would be tailored to specific functions we initiated an examination of the cross interface interactions of malate dehydrogenases from a wide variety of organisms whose crystal structures are available. Using the Hawkdock MM/GBSA routine we determined the residues involved in the interface in each structure. For the dimer interface we found between 24 and 35 residues involved depending on isoform. Mapping these residues onto structures revealed 5 areas of interaction, I to V with regions II – V being spatially conserved in all structures (figure 1). Region I was found only in chloroplastic versions of MDH. While the general areas of contact are conserved the detailed nature of the interactions revealed significant differences including inclusion of a conserved repulsion in region II in all Isoforms. These observations led to a series of hypotheses concerning the roles of specific regions and residues in isoform families. We focused on the cross interface interactions potentially related to citrate regulation of the organelle isoforms, and identified a unique cross interface interaction between the sidechain of D87 and the helix dipole of helix T268‐286 on the opposite subunit adjacent to a D92‐Y273 interaction across the interface. The role of L269/S266 in this helix and adjacent loop which we hypothesized form a cross interface molecular switch, was explored using site directed mutagenesis which showed that mutation of either residue to alanine disrupted either oxaloacetate or NADH saturation, overall catalytic activity and citrate inhibition. These results suggest a cross interface network (figure 2) in organelle malate dehydrogenases. Comparison of interface residues from structures with and without Citrate bound show that region III and V show the largest changes induced by citrate with some residues showing greater and others lesser contributions to the interface and are consistent with the observed network of interactions and suggest that changes across the interface might be induced by citrate interacting with S266. Interestingly the tetrameric MDHs from Ignacoccus Islandicus and the apicomplexans shares many but not all of these residues, lacking in particular D92, T268 and Y273. Knock in mutants of the Ignacoccus Islandicus and the Plasmodium falciparum MDHs studied both computationally and with enzyme kinetics shed further insight on the potential roles of these residues and the evolution of cooperativity in MDH.

Determination and Characterization of Genes that Encode the Nicotinate Dehydrogenase and 6‐Hydroxynicotinate Dehydrogenase Complexes within the Nicotinic Acid Degradation Pathway by Bacillus Niacini

N‐Heterocyclic aromatic compounds (NHACs) are constituents of many manufactured products such as pharmaceuticals, vitamins, and personal care products. Their widespread use has led to contamination of groundwater and soils, posing environmental concerns. Therefore, mechanisms of how NHACs are catabolized by bacteria have been studied to develop bioremediation strategies. Nicotinic acid (NA) is an example of an NHAC where its catabolism has been studied in a variety of microorganisms, such as Pseudomonas, Eubacterium,and Bacillus. While NA degradation has been elucidated in microorganisms such as P. putidaand E. barkeri,the majority of B. niacini’sNA catabolism pathway is relatively uncharacterized. Previous work identified a nicgene cluster that had been found to be upregulated in the presence of NA and 6‐hydroxynicotinic acid (6‐HNA), leading to the hypothesis that the set of protein subunits NicA1, NicA2, NicB1, NicB2 and CoxG are involved in the first two hydroxylation steps of B. niacini’sNA degradation pathway. These proteins are thought to form distinct multi‐subunit protein complexes with nicotinate dehydrogenase (NDH) and 6‐hydroxynicotinate dehydrogenase (6‐HNDH) activities. Due to the involvement of the molybdopterin cofactor present in the B subunits, recombinant forms of these enzymes have not shown activity when expressed and purified from E. coli. A vector specific for expressing the genes in Pseudomonas (pBBr) was engineered to contain the set of five B. niacini genes hypothesized to code for NA hydroxylating enzymes and was transformed into Pseudomonas fluorescens 1f2,a strain shown to be unable to degrade nicotinic acid naturally. Analysis of the media upon incubation of the Pf‐1f2 transformants with NA or 6‐HNA in minimal media by C‐18 chromatography (HPLC) showed rapid and complete hydroxylation of NA to 6‐HNA, as well as the hydroxylation of 6‐HNA to 2,6‐dihydroxynicotinic acid (2,6‐DHNA). This is the first evidence of activity observed from recombinant B. niacini“NicAB” protein complexes. However, the combination of gene‐encoded subunits that compose the NDH and 6‐HNDH complexes is currently unknown. Using deletion mutagenesis, specific genes cloned into the pBBr vector will be removed to recombinantly express variant complexes that lack a specific subunit to determine the substrate specificity and oligomeric structure of the complexes.

Developing a novel exenatide-based incretin mimic (αB-Ex): Expression, purification and structural-functional characterization

Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and exenatide have been well received in treating type 2 diabetes mellitus (T2DM) and obesity. In this study, an exenatide analogue, in which methionine at position 14 substituted with leucine, was ligated to human αB-crystallin (αB-Cry) and then expressed in the bacterial host cells. In the next step, the exenatide analogue was effectively released from the hybrid protein (αB-Ex) and subsequently purified using gel filtration chromatography. The HPLC and electrospray ionization mass spectrometry (ESI-MS) analyses respectively suggested a high purity (more than 97%) and an accurate molecular mass for the exenatide analogue (4168.22 Da and 835.01, z = 5). Also, the molecular mass of the αB-Ex hybrid protein based on the MALDI-TOF analysis was 24,702.162 Da. The secondary structure assessment by the three spectroscopic methods revealed that exenatide analogue and αB-Ex hybrid protein have an α-helix and a β-sheet rich structure, respectively. Also, according to the results of the DLS analysis, the αB-Ex hybrid protein indicated a high tendency to form large oligomeric structures. The NMR assessment suggested that the hybrid protein exists in its folding state. Both exenatide analogue and the αB-Ex hybrid protein revealed a crucial ability to reduce the blood sugar levels in healthy and diabetic mice. They were also capable of inducing insulin secretion to the bloodstream. Overall, our study introduces the αB-Ex hybrid protein as a novel incretin mimic, exerting its biological activity for a longer period of time. It might also be considered a potential drug candidate in the treatment of T2DM.

Antibacterial activity of the CAP18 peptide against Xanthomonas citri ssp. citri, the causative agent of citrus canker, as evaluated by in vitro and in silico studies

AbstractThe bacterium Xanthomonas citri ssp. citri (Xcc), causing citrus canker disease, poses a severe threat to citrus production worldwide. New approaches for managing the disease are required because of the limited number of bactericides available, reported pathogen resistance to copper pesticides, and the side effects of such compounds on citrus trees and the environment. In this study, the bacterium was isolated from a Mexican lime tree showing typical disease symptoms and identified as Xcc using conventional biochemical tests and specific PCR. The antibacterial activity of the recombinant peptide CAP18 (rCAP18) against the Xcc bacterium was assessed. Measuring the MIC parameter showed that purified rCAP18 caused bacterial growth inhibition by 90% at a concentration of 4.5 μM. Furthermore, a heterogeneous membrane system consisting of POPE:POPG:TOCL (5:1:3) was created using molecular dynamics simulation. The interaction effect of the oligomeric structure of rCAP18 (rCAP18OLIGO) on the components of a lipid bilayer membrane was determined by measuring the membrane thickness, the order parameter of the acyl chains and the area of each phospholipid. The results obtained from simulation and measurement of order parameters showed that, among the three phospholipids, rCAP18OLIGO more affected the orientation of carbon atoms in POPG. Fluctuations of average area per lipid for each phospholipid showed that rCAP18OLIGO had more interactions with POPG > TOCL > POPE, respectively, in the outer half of the membrane. The results obtained from in silico studies confirmed that the penetration of rCAP18OLIGO into the lipid bilayer membrane reduced the membrane thickness and caused its deformation during the simulation time. This mechanism is possibly the leading factor of Xcc growth inhibition by rCAP18 in vitro. Therefore, the rCAP18 peptide as an antibacterial agent can provide a way to develop a new approach to control the citrus canker disease.

Inhibition of Insulin Degrading Enzyme to Control Diabetes Mellitus and its Applications on some Other Chronic Disease: a Critical Review.

This review aims to provide a precise perceptive of the insulin-degrading enzyme (IDE) and its relationship to type 2 diabetes (T2D), Alzheimer's disease (AD), obesity, and cardiovascular diseases.The purpose of the current study wasto provideclearidea oftreating prevalent diseases such as T2D, and AD by molecular pharmacological therapeuticsrather than conventional medicinal therapy. To achieve the aims,molecular docking was performed using severalsoftwares such asLIGPLOT+, Python, and Protein-Ligand Interaction Profiler with corresponding tools. The IDE is a large zinc-metalloprotease that breakdown numerous pathophysiologically important extracellular substrates, comprising amyloid β-protein (Aβ) and insulin. Recent studies demonstrated that dysregulation of IDE leads to develop AD and T2D. Specifically, IDE regulates circulating insulin in a variety of organs via a degradation-dependent clearance mechanism. IDE is unique because it was subjected to allosteric activation and mediated via an oligomer structure. In this review, we summarised the factors that modulate insulin reformation by IDE and interaction of IDE and some recent reports on IDE inhibitors against AD and T2D. We also highlighted the latest signs of progress of the function of IDE and challenges in advancing IDE- targetted therapies against T2D and AD.

β-Lactoglobulin–fucoidan nanocomplexes: Energetics of formation, stability, and oligomeric structure of the bound protein

Interaction of β-lactoglobulin (BLG) with a sulfated polysaccharide isolated from the brown alga Fucus vesiculosus (fucoidan, FVF), was studied by nephelometry, high-sensitivity differential scanning calorimetry, and sedimentation velocity as a function of pH and the polysaccharide/protein ratio. FVF–BLG complexes were found over the pH 2.5–6.0 range. The complexation was accompanied by the decrease in the conformational stability of BLG at pH < pI 5.2 of the protein where FVF and BLG were oppositely charged. Near pI of the protein and at the pH values larger a bit than pI, where BLG has a large dipole moment or carries a small negative charge, the complexation appeared as changes in the oligomeric state of the protein. Binding curves of BLG to FVF were obtained at pH 3.0 and 6.0 according to the DSC and sedimentation velocity data. Binding constants were evaluated: 3.6 × 103 M−1 at pH 3.0 and 2 × 104 M−1 at pH 6.0. Correlations between the denaturation enthalpy and temperature for the free and bound protein in terms of Kirchhoff's law indicated that the denaturation of both forms of the protein is associated with comparable changes in the accessible surface area of the BLG molecule. It means that the binding of BLG to FVF does not perturb the hydrophobic core of the protein.

Nanoscale Structural Analysis of a Lipid-Driven Aggregation of Insulin.

Abrupt aggregation of misfolded proteins is a hallmark of a large number of severe pathologies, including diabetes types 1 and 2, Alzheimer, and Parkinson diseases. A growing body of evidence suggests that lipids can uniquely change rates of amyloid-associated proteins as well as modify the structure of formed oligomers and fibrils. In this study, we utilize atomic force microscopy infrared (AFM-IR) spectroscopy, also known as nano-IR spectroscopy, to examine the structure of individual insulin oligomers, protofilaments, and fibrils grown in the presence of phospholipids. Our findings show that AFM-IR spectra of insulin oligomers have strong signals of C-H and PO2- vibrations, which points on the presence of lipids in the oligomer structure. Furthermore, substantial shifts in lipid vibrations in AFM-IR spectra of the oligomers relative to the corresponding bands of pure lipids have been observed. This points on strong interactions between a lipid and a protein that are developed at the stage of the oligomer formation.

Cover Feature: A Hexagonal Shape‐Persistent Nanobelt of Elongated Rhombic Symmetry with Orthogonal π‐Planes by a One‐Pot Reaction (Eur. J. Org. Chem. 8/2022)

The Cover Feature shows the molecular structure (spacefill model; atoms of hydrogen are depicted in white, carbon in grey, nitrogen in blue, oxygen in bright red, and bromine in red) of the nanobelt synthesized by Lauer and colleagues. The nanobelt “swims” in a sea of many other oligomeric, polymeric, and monomeric structures and is “fished out” selectively by gel permeation chromatography, represented by a fishing hook. More information can be found in the Full Paper by M. Mastalerz et al.

The Pathological G51D Mutation in Alpha-Synuclein Oligomers Confers Distinct Structural Attributes and Cellular Toxicity.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure–function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Electro-mechanically switchable hydrocarbons based on [8]annulenes

Pure hydrocarbons with shape and conjugation properties that can be switched by external stimuli is an intriguing prospect in the design of new responsive materials and single-molecule electronics. Here, we develop an oligomeric [8]annulene-based material that combines a remarkably efficient topological switching upon redox changes with structural simplicity, stability, and straightforward synthesis: 5,12-alkyne linked dibenzo[a,e]cyclooctatetraenes (dbCOTs). Upon reduction, the structures accommodate a reversible reorganization from a pseudo-conjugated tub-shape to a conjugated aromatic system. This switching in oligomeric structures gives rise to multiple defined states that are deconvoluted by electrochemical, NMR, and optical methods. The combination of stable electromechanical responsivity and ability to relay electrons stepwise through an extended (pseudo-conjugated) π-system in partially reduced structures validate alkyne linked dbCOTs as a practical platform for developing new responsive materials and switches based on [8]annulene cores.

Metal-ion coordinated self-assembly of human insulin directs kinetics of insulin release as determined by preclinical SPECT/CT imaging

Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn2+ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe2+/Zn2+, Eu3+/Zn2+, Fe2+/Co3+) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the PheB1 or LysB29 position to create conjugates that bind either Fe2+ or Eu3+, while its natural binding site (HisB10) preferentially coordinates with either Zn2+ or Co3+. HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at PheB1 or LysB29, respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by 111In-radiolabeling for SPECT/CT imaging. When the 111In-B29Tpy-HI conjugate was coordinated with Fe2+/Zn2+, its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. 111In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded 111In-B1Tpy-HI was observed compared to that of 111In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., PheB1 or LysB29) on HI and its coordination with the added metal ions (i.e., Fe2+/Zn2+ or Fe2+/Co3+) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions.

Atomic view of an amyloid dodecamer exhibiting selective cellular toxic vulnerability in acute brain slices.

Atomic structures of amyloid oligomers that capture the neurodegenerative disease pathology are essential to understand disease-state causes and finding cures. Here we investigate the G6W mutation of the cytotoxic, hexameric amyloid model KV11. The mutation results into an asymmetric dodecamer composed of a pair of 30° twisted antiparallel β-sheets. The complete break between adjacent β-strands is unprecedented among amyloid fibril crystal structures and supports that our structure is an oligomer. The poor shape complementarity between mated sheets reveals an interior channel for binding lipids, suggesting that the toxicity may be due to a perturbation of lipid transport rather than a direct disruption of membrane integrity. Viability assays on mouse suprachiasmatic nucleus, anterior hypothalamus, and cerebral cortex demonstrated selective regional vulnerability consistent with Alzheimer's disease. Neuropeptides released from the brain slices may provide clues to how G6W initiates cellular injury.

Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the Saccharomyces cerevisiae During the Activation of UPR.

Nuclear degradation of pre-HAC1 mRNA and itssubsequent targeting plays a vital role in the activation as well as attenuation of Unfolded Protein Response (UPR) in Saccharomyces cerevisiae. Accurate measurement of the degradation of precursor HAC1 mRNA therefore appears vital to determine the phase of activation or attenuation of this important intracellular signaling pathway. Typically, pre-HAC1 mRNA degradation is measured by the transcription shut-off experiment in which RNA Polymerase II transcription is inhibited by a potent transcription inhibitor to prevent the de novo synthesis of all Polymerase II transcripts followed by the measurement of the steady-state levels of a specific (e.g., pre-HAC1) mRNA at different times after the inhibition of the transcription. The rate of the decay is subsequentlydetermined from the slope of the decay curve and is expressed as half-life (T1/2). Estimation of the half-life values and comparison of this parameter determined under different physiological cues (such as in absence or presence of redox/ER/heat stress) gives a good estimate of the stability of the mRNA under these conditions and helps gaining an insight into the mechanism of the biological process such as activation or attenuation of UPR.Intra-nuclear targeting of the pre-HAC1 mRNA from the site of its transcription to the site of non-canonical splicing, where the kinase-endonuclease Ire1p clusters into the oligomeric structures constitutes an importantaspectof the activation of Unfolded Protein Response pathway. These oligomeric structures are detectable as the Ire1p foci/spot in distinct locations across the nuclear-ER membrane under confocal micrograph using immunofluorescence procedure. Extent of the targeting of the pre-HAC1 mRNA is measurable in a quantified manner by co-expressing fluorescent-labeled pre-HAC1 mRNA and Ire1p protein followed by estimating their co-localization using FACS (Fluorescence-Activated Cell Sorter) analysis. Here, we describe detailed protocol of both determination of intra-nuclear decay rate and targeting-frequency of pre-HAC1 mRNA that were optimized in our laboratory.

Supramolecular structures of self-assembled oligomers under confinement.

We study the molecular origin of a prepeak (PP) observed at low q values in the structure factors of three oligomers in a bulk (poly(mercaptopropyl)methylsiloxane, PMMS, poly(methylmercaptopropyl)-grafted-hexylmethacrylate, PMMS-g-HMA, and poly(methylphenyl)siloxane, PMPS) in order to understand the lowering of the PP intensity detected for oligomers confined in cylindrical pores with low diameter. For this purpose, we use a combination of X-ray diffraction measurements and coarse-grained bead-spring molecular dynamics simulations. Our molecular modelling demonstrated that the planarity of the pendant groups triggers the self-association of oligomers into nanoaggregates. However, the formation of oligomeric nanodomains is not sufficient for building-up the PP. The latter requires spatial disturbance in the arrangement of the side groups of oligomers within clusters. Importantly, our numerical analysis revealed that the increasing degree of the confinement of oligomers limits their aggregation and consequently lowers the amplitude of the PP observed in the experimental data.

Computational Models for the Study of Protein Aggregation.

Protein aggregation has been studied by many groups around the world for many years because it can be the cause of a number of neurodegenerative diseases that have no effective treatment. Obtaining the structure of related fibrils and toxic oligomers, as well as describing the pathways and main factors that govern the self-organization process, is of paramount importance, but it is also very difficult. To solve this problem, experimental and computational methods are often combined to get the most out of each method. The effectiveness of the computational approach largely depends on the construction of a reasonable molecular model. Here we discussed different versions of the four most popular all-atom force fields AMBER, CHARMM, GROMOS, and OPLS, which have been developed for folded and intrinsically disordered proteins, or both. Continuous and discrete coarse-grained models, which were mainly used to study the kinetics of aggregation, are also summarized.

Mechanistic insight into the impact of a bivalent ligand on the structure and dynamics of a GPCR oligomer

Development of effective bivalent ligands has become the focus of intensive research toward modulation of G protein-coupled receptor (GPCR) oligomers, particularly in the field of GPCR pharmacology. Experimental studies have shown that they increased binding affinity and signaling potency compared to their monovalent counterparts, yet underlying molecular mechanism remains elusive. To address this, we performed accelerated molecular dynamics simulations on bivalent-ligand bound Adenosine 2A receptor (A2AR) dimer in the context of a modeled tetramer, which consists of A2AR and dopamine 2 receptor (D2R) homodimers and their cognate G proteins. Our results demonstrate that bivalent ligand impacted interactions between pharmacophore groups and ligand binding residues, thus modulating allosteric communication network and water channel formed within the receptor. Moreover, it also strengthens contacts between receptor and G protein, by modulating the volume of ligand binding pocket and intracellular domain of the receptor. Importantly, we showed that impact evoked by the bivalent ligand on A2AR dimer was also transmitted to apo D2R, which is part of the neighboring D2R dimer. To the best of our knowledge, this is the first study that provides a mechanistic insight into the impact of a bivalent ligand on dynamics of a GPCR oligomer. Consequently, this will pave the way for development of effective ligands for modulation of GPCR oligomers and hence treatment of crucial diseases such as Parkinson’s disease and cancer.

X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1: a novel helical oligomer structure.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.

The impact of lysyl-phosphatidylglycerol on the interaction of daptomycin with model membranes.

Daptomycin is an important clinical antibiotic for which resistance is rising. Daptomycin resistant strains of S. aureus often have increased 1,2-diacyl-sn-glycero-3-[phospho-1-(3-lysyl(1-glycerol))] (lysyl-PG) and mutations to the proteins directly involved in the synthesis and translocation of lysyl-PG are implicated in resistance mechanisms. To study the interaction of daptomycin with lysyl-DMPG-containing model membranes a new stereospecific and regioselective synthesis of lysyl-DMPG was developed. Studies on model membranes containing lysyl-DMPG demonstrate that: (1) daptomycin is not significantly repelled by the cationic charge of lysyl-DMPG; (2) daptomycin binds less avidly to lysyl-DMPG compared to DMPG; (3) the presence of lysyl-DMPG does not impact the membrane bound backbone conformation of daptomycin in a significant way; (4) lysyl-DMPG increases oligomer formation; (5) lysyl-DMPG does not impact model membrane fluidity at lysyl-PG : PG ratios that are relevant to daptomycin resistance. The results of these studies suggest that increased lysyl-PG content does not confer resistance to daptomycin by altering membrane fluidity or reducing membrane affinity but may confer resistance by altering the structure of daptomycin oligomers.

Amyloid management by chaperones: The mystery underlying protein oligomers' dual functions.

Protein oligomerization has two notable aspects: it is crucial for the performing cellular and molecular processes accurately, and it produces amyloid fibril precursors. Although a clear explanation for amyloidosis as a whole is lacking, most studies have emphasized the importance of protein misfolding followed by formation of cytotoxic oligomer structures, which are responsible for disorders as diverse as neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, and metabolic disorders, such as type 2 diabetes. Constant surveillance by oligomeric protein structures known as molecular chaperones enables cells to overcome the challenge of misfolded proteins and their harmful assemblies. These molecular chaperones encounter proteins in cells, and benefit cell survival as long as they perform correctly. Thus, this review highlights the roles of structural aspects of chaperone protein oligomers in determining cell fate-either succumbing to amyloid oligomers or survival-as well as experimental approaches used to investigate these entities.

Determination of the Most Stable Packing of Peptides from Ribosomal S1 Protein, Protein Bgl2p, and Aβ peptide in β-layers During Molecular Dynamics Simulations.

Our task was to determine the most stable packing of peptides in β-layers to construct an oligomer structure for fibril growth. The β-layers consisting of eight short peptides with the amino acid sequences IVRGVVVAID, VDSWNVLVAG (VESWNVLVAG), KLVFFAEDVG, and IIGLMVGGVV were built. These sequences correspond to the amyloidogenic regions of ribosomal S1 protein from E. coli, protein glucantransferase Bgl2p from the yeast cell wall, and Aβ peptide. First, the amyloidogenic regions were predicted theoretically, and then were confirmed experimentally. Four β-layers with different orientation of the peptides in the layers and the layers relative to each other were constructed. To determine the most stable packing of β-strands, the molecular dynamic (MD) simulations in explicit water were carried out. Two charge states (pH3 and pH5) for each β-layer were considered. The fraction of the secondary structure was a measure of stability for β-layers. β-Layers, in which β-strands are antiparallel relative to each other, were the most stable. Using this packing for β-strands, we constructed the oligomer structures and also checked their stability by using MD simulations.