Amino Acid Side Chains Research Articles

Exploring AlphaFold2's Performance on Predicting Amino Acid Side-Chain Conformations and Its Utility in Crystal Structure Determination of B318L Protein.

Recent technological breakthroughs in machine-learning-based AlphaFold2 (AF2) are pushing the prediction accuracy of protein structures to an unprecedented level that is on par with experimental structural quality. Despite its outstanding structural modeling capability, further experimental validations and performance assessments of AF2 predictions are still required, thus necessitating the development of integrative structural biology in synergy with both computational and experimental methods. Focusing on the B318L protein that plays an essential role in the African swine fever virus (ASFV) for viral replication, we experimentally demonstrate the high quality of the AF2 predicted model and its practical utility in crystal structural determination. Structural alignment implies that the AF2 model shares nearly the same atomic arrangement as the B318L crystal structure except for some flexible and disordered regions. More importantly, side-chain-based analysis at the individual residue level reveals that AF2's performance is likely dependent on the specific amino acid type and that hydrophobic residues tend to be more accurately predicted by AF2 than hydrophilic residues. Quantitative per-residue RMSD comparisons and further molecular replacement trials suggest that AF2 has a large potential to outperform other computational modeling methods in terms of structural determination. Additionally, it is numerically confirmed that the AF2 model is accurate enough so that it may well potentially withstand experimental data quality to a large extent for structural determination. Finally, an overall structural analysis and molecular docking simulation of the B318L protein are performed. Taken together, our study not only provides new insights into AF2's performance in predicting side-chain conformations but also sheds light upon the significance of AF2 in promoting crystal structural determination, especially when the experimental data quality of the protein crystal is poor.

Partitioning free energies and induced dipole response of small molecules across a POPC membrane using polarizable molecular dynamics.

The cell membrane is the semi-permeable barrier that regulates flux of materials in and out of the cell. The membrane has a characteristic dielectric gradient due to the amphiphilic nature of its constituent lipids, which have polar head groups and hydrophobic tails. This gradient plays governs many aspects of small-molecule partitioning and membrane protein folding and function. Atomistic details of small-molecule partitioning related to hydration and electronic polarization are still largely unknown. Here, we used polarizable molecular dynamics simulations to understand how the electronic environment influences the process of partitioning. We performed a series of unbiased and umbrella sampling (US) simulations using the Drude polarizable force field (FF) for six different amino-acid sidechain analogs in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. Using a polarizable FF such as Drude allows for the investigation of explicit dipole responses as a function of local electric fields along the membrane normal. We assessed molecular dipole moments, which vary as a function of localization within the membrane, and compared outcomes of the Drude FF simulations to those of the nonpolarizable CHARMM36 FF to investigate the differences in the free energy surfaces of partitioning for our different amino-acid analogs. In doing so, we sought to quantify the effects of using the fixed-charge convention of nonpolarizable FFs, which may average out the influence of the membrane dielectric gradient. We show how polarity of small molecules influences their partitioning and describe key differences in the free energy surfaces between polarizable and nonpolarizable systems in these contexts. This work serves as a foundation for future investigations into polarizable simulations of drug partitioning, as well as simulations of membrane proteins.

Molecular dynamics simulations suggest Thiosemicarbazones can bind p53 cancer mutant R175H

Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC – p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi‑sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.

Analysis of riboflavin/ultraviolet a corneal cross-linking by molecular spectroscopy

Corneal cross-linking (CXL) with riboflavin and ultraviolet A light is a therapeutic procedure to restore the mechanical stability of corneal tissue. The treatment method is applied to pathological tissue, such as keratoconus and induces the formation of new cross-links. At present, the molecular mechanisms of induced cross-linking are still not known exactly. In this study, we investigated molecular alterations within porcine cornea tissue after treatment with riboflavin and ultraviolet A light by surface enhanced Raman spectroscopy (SERS). For that purpose, after CXL treatment a thin silver layer was vapor-deposited onto cornea flaps. To explore molecular alterations induced by the photochemical process hierarchical cluster analysis (HCA) was used. The detailed analysis of SERS spectra reveals that there is no general change in collagen secondary structure while modifications on amino acid side chains are the most dominant outcome. The formation of secondary and aromatic amine groups as well as methylene and carbonyl groups were observed. Even though successful cross-linking could not be registered in all treated samples, Raman signals of newly formed chemical groups are already present in riboflavin only treated corneas.

Iron nutrition in agriculture: From synthetic chelates to biochelates

Iron (Fe) is an essential micronutrient for plant growth and productivity and, among other micronutrients, is the one required in higher amounts. In most soils, Fe occurs at relatively high concentrations; nevertheless, in calcareous soils, Fe bioavailability can be very limited, thus leading plants to develop Fe deficiency symptoms. At present, Fe fertilization still represents the most frequent approach adopted in agriculture to prevent or remediate Fe chlorosis. For soil applications, Fe fertilizers are based on Fe chelated with aminocarboxylate synthetic ligands (e.g. HEDTA, EDTA, DTPA, EDDHA), which are effective in maintaining Fe in the soil solution, even in alkaline soils, and thus in increasing its bioavailability for plant uptake. Nevertheless, Fe-aminocarboxylate complexes presents some limitations, related for instance to pH-dependent effectiveness, stability, persistence in the environment and ligand exchange reactions. In this context, vegetal-derived protein hydrolysates might represent good sources of biochelating ligands for developing innovative Fe fertilizers for sustainable agriculture. Protein hydrolysates are composed by small peptides, which, on one hand, can chelate Fe via amino acid side chains, carboxylate groups of C-termini, amine groups of N-termini and N atoms of amide groups of the peptide backbone, whilst, on the other hand, small peptides can also play a signaling role, triggering the acquisition of nutrient and morphogenetic processes in plants. The present paper reviews the current state of knowledge on traditional Fe fertilizers and, on the other side, explores the possible advantages in the application of biochelates as innovative Fe fertilizers. To further corroborate the hypothesis, three experimental trials have been carried out on three horticultural crops (cucumber, tomato, and strawberry) using a Fe-biochelate as Fe source and comparing it with the widely used traditional synthetic chelate Fe-EDDHA. The results in the three crops clearly show that Fe-biochelate is, at least, as efficient as Fe-EDDHA as source of micronutrient, even under circumneutral (pH 6.0) and alkaline conditions (pH 8.0), thus proving that Fe-biochelates can be promising alternatives to synthetic Fe chelates for the Fe nutrition management of crops. Considering the potential drawbacks of synthetic chelates (e.g., leachability, persistence, remobilization of toxic metals in soil), these findings might contribute enhancing the agriculture sustainability. In addition, Fe-biochelate could also find an application in soilless cultivation systems as an alternative to synthetic Fe chelates for Fe enrichment of edible plant tissues (biofortification), increasing their nutritional value.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

Inside-Out Design of Zinc-Binding Proteins with Non-Native Backbones.

The de novo design of functional proteins requires specification of tertiary structure and incorporation of molecular binding sites. Here, we develop an inside-out design strategy in the molecular modeling program Rosetta that begins with amino acid side chains from one or two α-helices making well-defined contacts with a ligand. A full-sized protein is then built around the ligand by adding additional helices that promote the formation of a protein core and allow additional contacts with the ligand. The protocol was tested by designing 12 zinc-binding proteins, each with 4-5 helices. Four of the designs were folded and bound to zinc with equilibrium dissociation constants varying between 95 nM and 1.1 μM. The design with the tightest affinity for zinc, N12, adopts a unique conformation in the folded state as assessed with nuclear magnetic resonance (NMR) and the design model closely matches (backbone root-mean-square deviation (RMSD) < 1 Å) an AlphaFold model of the sequence. Retrospective analysis with AlphaFold suggests that the sequences of many of the failed designs did not encode the desired tertiary packing.

Understanding the impact of halogen functional group (Br, Cl, F, OH) in amprenavir ligand of the HIV protease

We focused our attention towards the most dreadful disease that threatens the mankind of 20th century - Acquired immunodeficiency syndrome (AIDS), caused through the human immunodeficiency virus (HIV) and a sexually transmitted infection (STI). In this study, our foremost interest was to identify the potency and stability of HIV ligand- Amprenavir (APV) and its modelled functional group (Br, Cl, F, CF3, CH3, NH2) ligands through halogen and hydrogen bond contact, which will have a clear portrait on the structure activity of protein ligand interactions. This will assist chemist in synthesizing novel APV ligands, which are expected to inhibit the activity of HIV-1 protease enzyme. The binding strength of Amprenavir ligand with interacting hinge region amino acid side chains: Isoleucine (ILE 147, 150, 184), Valine (VAL 82), Alanine (ALA 28), Aspartic acid (25, 30, 125, 130) and Glycine (GLY 127, 149) were understood through interaction energy calculations at HF, B3LYP, M052X, MP2 level of theories for different basis set (6-311 G**, LANL2DZ). The present work will reveal an understandable picture about the halogen and hydrogen bond interaction that grip the contact of ligand and amino acids in the hinge region. Overall the Halogen atom (Br, Cl, F) functional groups improved the binding strength of APV in HIV protease; which provide a new novel path for the functional group preference on the ligand that enclose perfectly with the amino acid in the hinge region. Communicated by Ramaswamy H. Sarma

Substrate recognition mode of a glycoside hydrolase family 42 β-galactosidase from Bifidobacterium longum subspecies infantis (BiBga42A) revealed by crystallographic and mutational analyses.

Aim: Bifidobacterium longum subsp. infantis uses a glycoside hydrolase (GH) family 42 β-galactosidase (BiBga42A) for hydrolyzing lacto-N-tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, BiBga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants. Despite its importance, the structural basis underlying LNT hydrolysis by BiBga42A is not understood. Moreover, no substrate-complexed structures are available to date for GH42 family members. Methods: X-ray crystallography was used to determine the structures of BiBga42A in the apo- and liganded forms. The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-β-D-galactoside, lacto-N-biose I, LNT, and lacto-N-neotetraose (LNnT) as substrates. Conservation of those amino acid residues was examined among structure-determined GH42 β-galactosidases. Results: Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and catalysis in BiBga42A are conserved among all homologs including β-1,6-1,3-galactosidase (BlGal42A) from Bifidobacterium animalis subsp. lactis. Conclusion: BlGal42A is active on 3-β-galactobiose similarly to BiBga42A but is inactive on LNT. Interestingly, we found that the entrance of the catalytic pocket of BlGal42A is narrower than that of BiBga42A and seems not easily accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference may reflect the structural difference between the two enzymes.

Numerical 11-Factor in Half the Genetic Code – A Search for an Explanation in Number Series

Half of 20 + 4 (double-coded) amino acids in the genetic code structure showed1 an astonishing regularity in a 2D-table, including the 11-factor. The regularities have been dismissed by professionals, saying that no known physical-chemical laws can explain them. Here a search for possible explanations or aspects is carried through by investigating the numeral series, as dimensions, of x = integers 5 → 0 with different exponents, treated in earlier papers: x3, 2x2 behind the Periodic system, x2/3 ×102 (the “ES”-series) and further the basic series with its superposed series (as coding for underlying steps) with features like quantum mechanics. The regularities are reproduced in first section. It’s shown how the paired amino acid side chains that give the 11-factor in the 2D-table are derivable in different ways through counter-directed reading of numbers in the different series (2-digit numbers that always give sums with factor 11). Introduced is the concept of step-numbers, related to numeral series, implying a vector character, with this also anti-vectors, a physical and biological reality, expressed e.g. as intervals. A main interval in the ES-series as a whole is the number 77, specially studied, the most obvious organizing principle in the 2D-table. The fact that also sums of mirrored 2-letter codons code for amino acid groups with the 11-factor points to a deeper level of double-direction acting on both levels, here suggested as inherited from the higher dimension 4D. Regarding the coding base pairs G-C and A-U as two coordinate axes, the regularities show up in amino acids with 2-letter codons that join these axes, called Mx-coded ones, the other half of the genetic code not. It seems connected with the two axes of the 2D-table and strengthens the hypothetical dimensional processes from higher to lower D-degree. Finally, factor 9 as difference of numbers read bidirectionally are circa the inversion of 11, inversion existing in physical laws. An eventual relation to centriole structure with factor 9 (×2 or ×3) of microtubules are shortly studied. This paper should be taken as material, inviting other researchers in applied physics and biochemistry to go on with the questions.

A recombinant approach for stapled peptide discovery yields inhibitors of the RAD51 recombinase

An approach for stapled peptide preparation in small scale using recombinant expression of peptide–protein fusions in bacteria. We use this approach to design binders of RAD51, characterise their interaction and demonstrate activity in cells.

Dynamic geometry design of cyclic peptide architectures for RNA structure.

Designing inhibitors for RNA is still challenging due to the bottleneck of maintaining the binding interaction of inhibitor-RNA accompanied by subtle RNA flexibility. Thus, the current approach usually needs to screen thousands of candidate inhibitors for binding. Here, we propose a dynamic geometry design approach to enrich the hits with only a tiny pool of designed geometrically compatible scaffold candidates. First, our method uses graph-based tree decomposition to explore the complementarity rigid binding cyclic peptide and design the amino acid side chain length and charge to fit the RNA pocket. Then, we perform an energy-based dynamical network algorithm to optimize the inhibitor-RNA hydrogen bonds. Dynamic geometry-guided design yields successful inhibitors with low micromolar binding affinity scaffolds and experimentally competes with the natural RNA chaperone. The results indicate that the dynamic geometry method yields higher efficiency and accuracy than traditional methods. The strategy could be further optimized to design the length and chirality by adopting nonstandard amino acids and facilitating RNA engineering for biological or medical applications.

A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model.

Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.

Peptide-based porous materials and their applications

The porous structure formed by the self-assembly of peptides is a new generation of porous materials with potential applications in the fields of catalysis, sensing, separation, and drug delivery. The pores of the material are filled with amino acid side chains, so the pore size and properties of the peptide-based porous material can be adjusted by rational design or modification of the peptide sequences. The introduction of functional moieties and metal ions has expanded the range of peptide-based porous materials. This review covers the design, synthesis, self-assembly, and properties of peptide-based porous materials, and summarizes their applications in different fields in recent years.

Ensemble Modified Aptamer Based Pattern Recognition for Adaptive Target Identification.

The difficulty of the molecular design and chemical synthesis of artificial sensing receptors restricts their diagnostic and proteomic applications. Herein, we report a concept of "ensemble modified aptamers" (EMAmers) that exploits the collective recognition abilities of a small set of protein-like side-chain-modified nucleic acid ligands for discriminative identification of molecular or cellular targets. Different types and numbers of hydrophobic functional groups were incorporated at designated positions on nucleic acid scaffolds to mimic amino acid side chains. We successfully assayed 18 EMAmer probes with differential binding affinities to seven proteins. We constructed an EMAmer-based chemical nose sensor and demonstrated its application in blinded unknown protein identification, giving a 92.9% accuracy. Additionally, the sensor is generalizable to the detection of blinded unknown bacterial and cellular samples, which enabled identification accuracies of 96.3% and 94.8%, respectively. This sensing platform offers a discriminative means for adaptive target identification and holds great potential for diverse applications.

Protein digestion and absorption: the influence of food processing.

The rates of dietary protein digestion and absorption can be significantly increased or decreased by food processing treatments such as heating, gelling and enzymatic hydrolysis, with subsequent metabolic impacts, e.g. on muscle synthesis and glucose homeostasis.This review examines in vivo evidence that industrial and domestic food processing modify the kinetics of amino acid release and absorption following a protein-rich meal. It focuses on studies that used compositionally-matched test meals processed in different ways.Food processing at extremely high temperature at alkaline pH and/or in the presence of reducing sugars can modify amino acid sidechains, leading to loss of bioavailability. Some protein-rich food ingredients are deliberately aggregated, gelled or hydrolysed during manufacture. Hydrolysis accelerates protein digestion/absorption and increases splanchnic utilisation. Aggregation and gelation may slow or accelerate proteolysis in the gut, depending on the aggregate/gel microstructure.Milk, beef and eggs are heat processed prior to consumption to eliminate pathogens and improve palatability. The temperature and time of heating affect protein digestion and absorption rates, and effects are sometimes non-linear. In light of a dietary transition away from animal proteins, more research is needed on how food processing affects digestion and absorption of non-animal proteins.Food processing modifies the microstructure of protein-rich foods, and thereby alters protein digestion and absorption kinetics in the stomach and small intestine. Exploiting this principle to optimise metabolic outcomes requires more human clinical trials in which amino acid absorption rates are measured and food microstructure is explicitly considered, measured and manipulated.

Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry.

ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

Cumulative Millisecond-Long Sampling for a Comprehensive Energetic Evaluation of Aqueous Ionic Liquid Effects on Amino Acid Interactions

The interactions of amino acid side-chains confer diverse energetic contributions and physical properties to a protein's stability and function. Various computational tools estimate the effect of changing a given amino acid on the protein's stability based on parametrized (free) energy functions. When parametrized for the prediction of protein stability in water, such energy functions can lead to suboptimal results for other solvents, such as ionic liquids (IL), aqueous ionic liquids (aIL), or salt solutions. However, to our knowledge, no comprehensive data are available describing the energetic effects of aIL on intramolecular protein interactions. Here, we present the most comprehensive set of potential of mean force (PMF) profiles of pairwise protein-residue interactions to date, covering 50 relevant interactions in water, the two biotechnologically relevant aIL [BMIM/Cl] and [BMIM/TfO], and [Na/Cl]. These results are based on a cumulated simulation time of >1 ms. aIL and salt ions can weaken, but also strengthen, specific residue interactions by more than 3 kcal mol-1, depending on the residue pair, residue-residue configuration, participating ions, and concentration, necessitating considering such interactions specifically. These changes originate from a complex interplay of competitive or cooperative noncovalent ion-residue interactions, changes in solvent structural dynamics, or unspecific charge screening effects and occur at the contact distance but also at larger, solvent-separated distances. This data provide explanations at the atomistic and energetic levels for complex IL effects on protein stability and should help improve the prediction accuracies of computational tools that estimate protein stability based on (free) energy functions.

Recent Progress in Biosensors for Diagnosis of Cancer Cells, Tissues and Tumors Biomarkers

Raman spectroscopy is an important method for identifying molecules, which is widely used in determining the chemical and structural characteristics of various substances. Many materials have a special Raman spectrum, so that this phenomenon has turned the Raman device into an efficient tool for studying the structural and chemical properties of molecules. Since it is possible to obtain detailed information about the chemical and structural characteristics of biological compounds from Raman spectroscopy, the use of this method is rapidly expanding in the field of life sciences, especially in biological and medical studies. There is no need for special, time-consuming and expensive preparations in the study of materials with the help of a Raman device. In the protein Raman spectrum, distinct bands arise from the vibrational states of the peptide backbone and amino acid side chains. Therefore, based on the position and intensity of the protein’s Raman spectrum, it is possible to obtain valuable information about its second, third, and fourth structures. Also, the Raman spectrum of the protein contains information about the orientation and surrounding environment of the amino acid side chains. The correct formation of the disulfide bond in the protein structure can also be studied with the help of the Raman device. In general, the Raman spectrum of proteins contains multiple discrete bands that represent the vibrational states of the molecule and is used as a selective fingerprint to accurately determine the three–dimensional structure of proteins, intramolecular dynamics, and intermolecular interactions (Graphical Abstract).

Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting.

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.