Chain Residues Research Articles

Structural insights into epitope-paratope interactions of a monoclonal antibody targeting CEACAM5-expressing tumors

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are overexpressed in some tumor types. The antibody-drug conjugate tusamitamab ravtansine specifically recognizes the A3-B3 domains of human CEACAM5 (hCEACAM5). To understand this specificity, here we map the epitope-paratope interface between the A3-B3 domains of hCEACAM5 (hCEACAM5A3-B3) and the antigen-binding fragment of tusamitamab (tusa Fab). We use hydrogen/deuterium exchange mass spectrometry to identify the tusa Fab paratope, which involves heavy chain (HC) residues 101–109 and light chain residues 48–54 and 88–104. Using surface plasmon resonance, we demonstrate that alanine variants of HC residues 96–108 abolish binding to hCEACAM5, suggesting that these residues are critical for tusa-Fab–antigen complex formation. The cryogenic electron microscopy structure of the hCEACAM5A3-B3- tusa Fab complex (3.11 Å overall resolution) reveals a discontinuous epitope involving residues in the A3-B3 domains and an N-linked mannose at residue Asn612. Conformational constraints on the epitope-paratope interface enable tusamitamab to target hCEACAM5A3-B3 and distinguish CEACAM5 from other CEACAMs.

2-Methoxy-4-formylphenol suppresses methylglyoxal glycation mediated structural alterations and esterase activity of hemoglobin – A multi spectroscopic, biophysical and in-silico study

Glycation is the non-enzymatic reaction of glucose or its metabolites to proteins, causing irreversible changes. Methylglyoxal, a dicarbonyl, affects the structure and function of physiologically important proteins. Being a major circulatory protein, hemoglobin is highly prone to glycation. Current research focuses on identifying potent glycation inhibitors to prevent glycation and their impact on protein structure and function. The present study investigates the Advanced Glycation Endproducts (AGEs) inhibitory effects of 2-methoxy-4-formylphenol (Vanillin) against methylglyoxal mediated glycation of hemoglobin. The hemoglobin-vanillin glycation model exhibited inhibition of AGE formation, amyloid fibrils, aggregates and reduction in esterase activity. The fluorescence spectroscopic technique revealed efficient binding of vanillin and hemoglobin, with Stern Volmer plot indicating the presence of static quenching. The conformational stability of the vanillin and hemoglobin interaction was also evident from the molecular docking and dynamics studies. The proximal orientation of residues (H2 and K82 associated in esterase activity) of hemoglobin β1 chain and vanillin, supports the noted effect of reduced esterase activity in the presence of vanillin in glycated hemoglobin and the inhibition of the overall formation of AGE of hemoglobin in the presence of vanillin.

Site-directed mutagenesis leads to the optimized transglycosylation activity of endo-beta-N-acetylglucosaminidase from Trypanosoma brucei.

Endo-β-N-acetylglucosaminidases (ENGases) are pivotal enzymes in the degradation and remodeling of glycoproteins, which catalyze the cleavage or formation of β-1,4-glycosidic bond between two N-acetylglucosamine (GlcNAc) residues in N-linked glycan chains. It was investigated that targeted mutations of amino acids in ENGases active site may modulate their hydrolytic and transglycosylation activities. Endo-Tb, the ENGase derived from Trypanosoma brucei, belongs to the glycoside hydrolase family 85 (GH85). Our group previously demonstrated that Endo-Tb exhibits hydrolytic activity toward high-mannose and complex type N-glycans and preliminarily confirmed its transglycosylation potential. In this study, we further optimized the transglycosylation activity of recombinant Endo-Tb by focusing on the N536A, E538A and Y576F mutants. A comparative analysis of their transglycosylation activity with that of the wild-type enzyme revealed that all mutants exhibited enhanced transglycosylation capacity. The N536A mutant exhibited the most pronounced improvement in transglycosylation activity with a significant reduction in hydrolytic activity. It is suggested that Endo-Tb N536A possesses the potential as a tool for synthesizing a wide array of glycoconjugates bearing high-mannose and complex type N-glycans.

Furan-containing polymer-coated magnetic nanoparticles using the ‘graft-to’ approach

Coating of magnetic nanoparticles with functional polymers increases the applicability of these nanomaterials. A modular approach to functionalizing these polymer coatings can be achieved using click chemistry. This work reports the synthesis of magnetic nanoparticles coated with a dopamine anchoring group containing polymer with furan groups as side chain residues. Using the ‘graft-to’ approach, polymers are coated onto magnetic nanoparticles using a simple place exchange reaction. Various analytical techniques were used to characterize the polymer-coated nanoparticles for their size and surface composition. Utilization of poly(ethylene glycol) (PEG)-based polymer renders the magnetic nanoparticles water dispersible. The presence of the furan ring on the polymer brushes enables their facile functionalization using a maleimide group-bearing molecule under catalyst-free conditions. A hydrophobic fluorescent dye, used as a model drug, is conjugated onto the nanoparticles using the Diels-Alder reaction. One can anticipate that the facile fabrication and functionalization of these clickable polymer-coated magnetic nanoparticles will be attractive for various biomedical applications.

Lignin-Stabilized Tough, Sticky, and Recyclable Liquid Metal/Protein Organogels for Multifunctional Epidermal Smart Materials.

Biomass-encapsulated liquid metals (LMs) composite gels have aroused tremendous attention as epidermal smart materials due to their biocompatibility and sustainability. However, they can still not simultaneously possess toughness, adhesion, and recoverability. In this work, the tough, sticky, and recyclable protein-encapsulated LMs organogels (GLMx) are fabricated through the micro-interfacial stabilization of LMs by lignin and the following preparation of food-making inspired gels. With the help of lignin modification, the LMs micro-drops demonstrated uniform dispersion in the protein matrix, as well as dense non-covalent interactions (e.g., H─bond and hydrophobic interaction) with amino acid residues in peptide chains, which endowed the GLMx with high conductivity (≈5.4Sm-1), toughness (≈738.2kJm-3), self-adhesiveness (a maximal lap-shear strength of ≈58.3kPa), and recoverability. By tightly adhering onto human skin, the GLMx can act as epidermal sensors to detect drastic (e.g., joint bending) and subtle body movements (e.g., swallowing) and even recognize handwriting and speaking in real-time. Moreover, the organogels can also harvest solar energy and convert it into heat and electricity, which is promising in self-powered intelligent devices. Thus, this work paves a facile way to prepare protein/LMs composite organogels that are suitable for multiple applications like healthcare, human-robot interactions, and solar energy conversion.

The Bifidobacterium adolescentis BAD_1527 gene encodes GH43_22 α-L-arabinofuranosidase of AXH-m type

Bifidobacterium adolescentis gene BAD_1527 has previously been suggested to code for a β-xylosidase (Kobayashi et al., Mar Drugs 18:174, 2020). Our detailed investigation of the substrate specificity of the GH43_22 protein using a wide spectrum of natural and artificial substrates showed that the enzyme hydrolyzed neither linear xylooligosaccharides nor glucuronoxylan. Xylose was released only from the artificial 4-nitrophenyl β-D-xylopyranoside (1.58 mU/mg). The corresponding α-L-arabinofuranoside was by three orders of magnitude better substrate (2.17 U/mg). Arabinose was the only monosaccharide liberated from arabinoxylan and α-1,3- or α-1,2-singly arabinosylated xylooligosaccharides. Moreover, the enzyme efficiently debranched sugar beet arabinan and singly arabinosylated α-1,5-L-arabinooligosaccharides, although short linear α-1,5-L-arabinooligosaccharides were also slowly degraded. On the other hand, debranched arabinan, arabinogalactan as well as 2,3-doubly arabinosylated main chain residues of arabinan and arabinoxylan did not serve as substrates. Thus, the enzyme encoded by the BAD_1527 gene is a typical α-L-arabinofuranosidase of AXH-m specificity.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS)

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[β-D-Xylp-(1 → 3)]-D-Manp-(1 → 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 → 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2–3)-α-L-Rhap-(1 → 3)]-D-Manp-(1 → 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 → 3)-β-D-Glcp-(1 →. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Self-healing amino acid-bearing acrylamides/n-butyl acrylate copolymers via multiple noncovalent bonds.

Four amino acid-bearing acrylamides, N-acryloyl-l-threonine (AThrOH), N-acryloyl-l-glutamic acid (AGluOH), N-acryloyl-l-phenylalanine (APheOH), and N-acryloyl-l, l-diphenylalanine (APhePheOH), were selected for copolymerization with n-butyl acrylate (nBA) to develop amino acid-based self-healable copolymers. A series of copolymers comprising amino acid-bearing acrylamides and nBA with tunable comonomer compositions and molecular weights were synthesized by free radical and reversible addition-fragmentation chain-transfer copolymerization. Self-healing and mechanical properties originated from the noncovalent bonds between the carboxyl, hydroxyl, and amide groups, and π-π stacking interactions among the amino acid residues in the side chains were evaluated. Among these copolymers, P(nBA-co-AGluOH) with suitable comonomer compositions and molecular weights (nBA : AGluOH = 82 : 18, Mn = 18 300, Mw/Mn = 2.58) exhibited good mechanical properties (modulus of toughness = 17.3 MJ m-3) and self-healing under ambient conditions. The multiple noncovalent bonds of P(nBA-co-AGluOH)s were also efficient in improving the optical properties with an enhanced refractive index and good transparency.

Synthesis of trehalose-grafted poly(ester amide) with potential ice recrystallization inhibition activity

The ice recrystallization inhibition (IRI) activity of a macromolecular cryoprotectant is of great significance for applications in cryopreservation of biosamples. Herein, in order to create a possibly biodegradable trehalose-modified polymer, thiol-monosubstituted trehalose was synthesized by a simplified method, and then tethered to allylated-based poly(ester amide) (PEA) via thiol-ene click reaction. The synthesized PEA-g-trehalose (PEA-g-Tre) was able to inhibit ice recrystallization by 55% at 10 mg mL−1 compared to PBS control, superior to poly(ethylene glycol). Differential scanning calorimetry results showed that PEA-g-Tre exhibited a significantly higher glass transition temperature at 92.4 °C than PEA at 33.5 °C, and had a narrowed ice crystallization temperature range due to introduction of trehalose. Raman spectroscopy indicated that the IRI activity of PEA-g-Tre was attributed to the presence of trehalose residues in the side groups, which disrupted the regular arrangement of freezable water molecules. It is suggested that modification by trehalose can give rise to the IRI activity of the polymer through adsorption and diffusion mechanism with water, and thiol-ene reaction provides a feasible way for preparing polymeric cryoprotectants containing trehalose.

Molecular Insights into the Covalent Binding of Zoxamide to the β-Tubulin of Botrytis cinerea.

Botrytis cinerea is a fungal plant pathogen that causes significant economic losses in the agricultural industry worldwide. Fungicides that target microtubules, such as carbendazim (CBZ), diethofencarb (DEF), and zoxamide (ZOX), are widely used in crop protection against this pathogen. These groups of compounds exert their fungicidal activity by disrupting the microtubule assembly by binding to the β-tubulin subunit, provoking cell-cycle arrest and cell death. However, with the appearance of isolates resistant to these compounds, it is necessary to search for new alternatives to control this pathogenic fungus. In this work, we gained insight into the binding and stability of these fungicides in the benzimidazole binding site of B. cinerea β-tubulin through different computational approaches. Our molecular dynamics simulation replicas showed that R enantiomers of ZOX and its analog RH-4032 had better interaction profiles at the site compared to S enantiomers. The simulations also revealed that while the R-isomer fungicides formed H-bonds with the main chain carbonyl of V236 or the side chain residue of S314, only CBZ interacted with E198. Previous experimental data have identified key mutations in B. cinerea's β-tubulin gene that lead to the development of resistance or, on the contrary, increased sensitivity for treatment with these fungicide compounds. In agreement with experimental findings, alchemical free energy calculations showed that E198A and E198V mutations in B. cinerea β-tubulin have high sensitivity to (R)-ZOX, whereas the E198K mutation decreased its affinity. Similarly, the results obtained explain the resistance to CBZ of B. cinerea isolates with E198A/V/K mutations and the insensitivity of the wild-type organism to DEF. Our work provides a deeper insight into the molecular mechanism of action of these fungicides, highlighting the importance of understanding the interaction profiles to develop more effective antifungal agents.

Determining glass transition in all-atom acrylic polymeric melt simulations using machine learning.

The functionality of many polymeric materials depends on their glass transition temperatures (Tg). In computer simulations, Tg is often calculated from the gradual change in macroscopic properties. Precise determination of this change depends on the fitting protocols. We previously proposed a robust data-driven approach to determine Tg from the molecular dynamics simulation data of a coarse-grained semiflexible polymer model. In contrast to the global macroscopic properties, our method relies on high-resolution microscopic details. Here, we demonstrate the generality of our approach by using various dimensionality reduction and clustering methods and apply it to an atomistic model of acrylic polymers. Our study reveals the explicit contribution of the side chain and backbone residues in influencing the determination of the glass transition temperature.

Structural Insights into Protein-Ligand Interactions of Small Leucine Rich Repeat Proteoglycans with a Large Number of Binding Partners: An Overview

Small leucine rich repeat proteoglycans (SLRPs) exist in the extracellular matrix. SLRPs contain tandem arrays of LRRs flanked by cysteine clusters at the both N- and C-termini. The extreme N- and/or C-termini contain low complexity sequences, glycosaminoglycan (GAG) chain and/or sulfated tyrosine residues in some members of SLRPs. The LRR solenoid structure may be divided into four parts consisting of a concave surface, an ascending surface, a convex surface, and a descending surface. SLRPs share many biological functions including collagen fibrillogenesis, signaling, innate immunity, and inflammation through the binding of various ligands. We undertake a comprehensive literature search of publications in order to make a list of ligands of SLRPs. We describe and discuss the interacting sites of SLRPs to binding partners. The protein-ligand interactions occur on not only the concave surface but also the ascending surface and the N- or C-terminal capping regions. In addition, the extreme N- and/or C-terminal regions with the GAG chains or sulfated tyrosine residues participate in ligand-interactions.

Multiple Non-Canonical Base-Stacking Interactions as One of the Major Determinants of RNA Tertiary Structure Organization.

Stacking interactions of heterocyclic bases of ribonucleotides are one of the most important factors in the organization of RNA secondary and tertiary structure. Most of these (canonical) interactions are formed between adjacent residues in RNA polynucleotide chains. However, with the accumulation of data on the atomic tertiary structures of various RNAs and their complexes with proteins, it has become clear that nucleotide residues that are not adjacent in the polynucleotide chains and are sometimes separated in the RNA primary structure by tens or hundreds of nucleotides can interact via (non-canonical) base stacking. This paper presents an exhaustive database of such nonadjacent base-stacking elements (NA-BSEs) and their environment in the macromolecules of natural and synthetic RNAs. Analysis of these data showed that NA-BSE-forming nucleotides, on average, account for about a quarter of all nucleotides in a particular RNA and, therefore, should be considered as bona fide motifs of the RNA tertiary structure. We also classified NA-BSEs by their location in RNA macromolecules. It was shown that the structure-forming role of NA-BSEs involves compact folding of single-stranded RNA loops, transformation of double-stranded bulges into imperfect helices, and binding of RNA regions distant in the primary and secondary RNA structure.

Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules

Abstract Human leucocyte antigens (HLAs) display a repertoire of epitopic peptides on the cell surface for T cell recognition. Amino acid polymorphisms in the peptide-binding groove of different HLA allotypes result in distinct antigenic repertoires, providing a basis for adaptive immunity to emerging pathogens at the population level. T cell receptors (TCRs) recognize pHLA antigens through interactions with both peptide and heavy chain residues. In this work, we use structural data from peptide:MHC-I and pMHC:TCR structures to identify residues important for peptide and/or TCR binding, and we outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Moreover, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules displaying tumor-associated antigens with modified TCR interaction surfaces, which could further serve as platforms to elicit alloreactive T cell responses against self-antigens that are overexpressed in cancer. Finally, we gain a deeper understanding of the evolution and functional divergence of the different HLA allotypes by demonstrating an example of convergent evolution. Supported by grants from NIAID (5R01AI143997), NIGMS (5R35GM125034), and NIDDK (5U01DK112217)

All-atom simulations of the trimeric spike protein of SARS-CoV-2 in aqueous medium: Nature of interactions, conformational stability and free energy diagrams for conformational transition of the protein.

The spike protein of SARS-CoV-2 exists in two major conformational states, namely the 'open' and 'closed' states which are also known as the 'up' and 'down' states, respectively. In its open state, the receptor binding domain (RBD) of the protein is exposed for binding with ACE2, whereas the spike RBD is inaccessible to ACE2 in the closed state of the protein. In the current work, we have performed all-atom microsecond simulations of the full-length trimeric spike protein solvated in explicit aqueous medium with an average system size of ~0.7 million atoms to understand the molecular nature of intra- and inter-chain interactions, water-bridged interactions between different residues that contribute to the stability of the open and closed states of the protein, and also the free energy landscape for transition between the open and closed states of the protein. We have also examined the changes of such interactions that are associated with switching from one state to the other through both unbiased and biased simulations at all-atom level with total run length of 4μs. Interestingly, after about 0.8μs of unbiased molecular dynamics run of the spike system in the open state, we observed a gradual transition of the monomeric chain (B) from open to its partially closed or down state. Initially the residues at the interface of chain B RBD in the open state spike protein were at non-hydrogen-bonding distances from the residues of chain C RBD. However, the two RBDs gradually came closer and finally the residue S459 of the RBD of chain B made a hydrogen bond with F374 of chain C in the last 200 ns of the simulation along with formation of a few more hydrogen bonds involving other residues. Since no transition from closed to the open state of the protein is observed in the present 1μs unbiased simulation of the closed state protein, the current study seems to suggest that the closed conformational state is preferred for the spike protein of SARS-CoV-2 in aqueous medium. Furthermore, calculations of the free energy surface of the conformational transition from open (up) to the closed (down) state using a biased simulation method reveal a free energy barrier of ~3.20 kcal/mol for the transition of RBD from open to the closed state, whereas the barrier for the reverse process is found to be significantly higher.

A Novel Epitope Dataset: Performance of the MCL-Based Algorithms to Generate Dataset for Graph Learning Model

Naturally, the epitope dataset can be presented as a graph. Dataset preparation in the previous methods is part of model development. There are many graph-based classification and regression methods. Still, it is difficult to identify their performance on the conformational epitope prediction model because datasets in a suitable format are unavailable. This research aims to build a dataset in a suitable format to evaluate kernel graph and graph convolution network. This dataset, which results from graph clustering on graph antigens, can be used to identify the performance of many graph neural network-based algorithms for conformational epitope prediction. The Ag-Ab complexes that meet the criteria for forming a conformational epitope prediction dataset from previous studies were downloaded from the Protein Data Bank. Raw datasets in the form of specific exposed antigen chain residues are labeled as epitope or non-epitope based on their proximity to the paratope. The engineering features in the raw dataset are derived from the structure of the antigen-antibody complex and the propensity score. Aggregating atomic-level interactions into residual levels create an initial graph of the antigen chain. The MCL, MLR-MCL, and PS-MCL are graph clustering algorithms to obtain labeled sub-clusters from the initial graph. A balance factor parameter is set to several values to identify the optimal dataset formation based on minimal fragmentation. The output of the MCL algorithm is used as a baseline. As a result of the fragmentation analysis that occurs, the MLR-MCL algorithm gives the best model performance at a balance factor equal to 2. PS-MCL gives the best performance at a value of 0.9. Based on the minimum fragmentation, the MLR-MCL algorithm provides the best model performance compared to MCL and PS-MCL. The dataset in a format according to benchmarking dataset can be used to identify the characteristics of antigen subgraphs formed from the graph clustering process and to explore the performance of graph-based learning conformational epitope prediction models such as graph convolution networks.

Structural insights into substrate accommodation by Escherichia coli adenine phosphoribosyltransferase (APRT) reveal a unique mechanism in enterobacterales.

Nucleotide biosynthesis is one of the vital mechanisms of living organisms. The salvage pathway of nucleotide synthesis governs an energy efficient route, utilizes the intermediates from the degradation pathway and provides the swift supply of nucleotides needed for essential cellular activities. The adenine phosphoribosyltransferase (APRT) is a part of the purine salvage pathway enzyme that catalyzes the transfer of the phosphoribosyl moiety from 5-phosphoribosyl 1-pyrophosphate (PRPP) to free nucleotide bases. Though this enzyme is well characterized across various taxonomical orders still the details of substrate specificity, enzyme activity and its physiological role in survival are still unexplored in Enterobacterales. This group comprised species that form the largest group of bacterial pathogens along with being the part of gastrointestinal tract microflora in humans. Therefore, a thorough investigation of the function of Enterobacterales's APRT and identification of a novel mechanism may aid in the development of new antibiotics to prevent the spread of multidrug resistance strains of the Enterobacterales. In order to delineate molecular mechanism of the substrate binding, we determined the high-resolution crystal structure of E. coli APRT (EcAPRT) in both apo and adenine bound forms. The EcAPRT enzyme has a core PRPP binding domain and a distinct auxiliary region comprising an N-terminal hood and flexible loop. The binding of adenine in EcAPRT is facilitated through an interesting mechanism in which the side chain conformation of carboxy-terminal residue flips and a substrate trap is generated by the side chain residues.

On the electrochemical oxidation of methionine residues of proteins

The oxidation of methionine side chain residues in proteins provides a great diversity of possible oxidation mechanisms dictated, among others, by the oxidizing species, solvent properties, and protein structure. The oxidation behavior of a series of short acetylated synthetic peptides, Ac-GMG, Ac-GGMGG and Ac-GGGMGGG, was investigated by differential pulse and square wave voltammetry, in a wide pH range, at glassy carbon electrode. It was found that always the first oxidation step represents the one-electron oxidation of thioether moiety with the formation of a radical cation. Following this, depending on the experimental conditions and side chain position of methionine, the radical is stabilized by the nucleophilic attack of water or by catalytic support of the neighboring carbonyl and amide groups in an intermediate structure, finally converted in methionine sulfoxide which can be further oxidized, at more positive potential, into methionine sulfone. For Ac-GGGMGGG, at pH 8.0, the amino and amide groups are active involved in the oxidation process and the electrode reaction takes place with proton transfer

Early-Stage Oligomerization of Prion-like Polypeptides Reveals the Molecular Mechanism of Amyloid-Disrupting Capacity by Proline Residues.

Proline cis/trans isomerization governs protein local conformational changes via its local mechanical rigidity. The amyloid-disrupting capacity of proline is widely acknowledged; however, the molecular mechanism is still not clear. To understand how proline residues in polypeptide chains influence amyloid propensity, we study several truncated sequences of the TDP-43 C-terminal region (287-322) and their triple proline variants (308PPP310). We use coarse-grained molecular simulation to study the time evolution of the process of aggregation in the early stages in an effective high-concentration condition (∼25 mM). This ensures the long time scales for protein association at laboratory concentrations. We use several experimentally determined structure templates as initial structures of monomer conformations. We carry out oligomer size analysis and cluster analysis, along with several structural measures, to characterize the size distributions of oligomers and their morphological/structural properties. We show that average oligomer size is not a good indicator of amyloid propensity. Structural order and/or morphological properties are better alternatives. We show that proline variants can efficiently maintain the formation of large "ordered" oligomers of shorter truncated sequences, i.e., 307-322. This "order" maintenance is weakened when using longer truncated sequences (i.e., 287-322), leading to the formation of "disordered" oligomers. From an energy trade-off perspective, if the entropic effect is weak (short sequence length), the shape-complementarity of proline variants effectively guides the oligomerization process to form "ordered" oligomer intermediates. This leads to a distinct aggregation pathway that promotes amyloid formation (on-pathway). Strong entropic effects (long sequence length), however, would cause the formation of "disordered" oligomers. This in turn will suppress amyloid formation (off-pathway). The proline shape-complementary effects provide a guided morphological restraint to facilitate the pathways of amyloid formation. Our study supports the importance of structure-based kinetic heterogeneity of prion-like sequence fragments in driving different aggregation pathways. This work sheds light on the role of morphological and structural order of early-stage oligomeric species in regulating amyloid-disrupting capacity by prolines.