Protonated Amino Acids Research Articles

Detection of Neutral CO Lost During Ionic Dissociation Using Atmospheric Pressure Thermal Dissociation Mass Spectrometry (APTD-MS).

Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., + 1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus. Graphical Abstract ᅟ.

Articular Cartilage: The Destruction of Bilayers and Deactivation of Phospholipid Molecules

To our knowleadge the coaugulation by interacton of β2 -Glycoprotein I (β2 -GP I) with the phospholipid membrane is required to verify this hypothesis. The open hockey stick-like conformation occurs when protonated amino acid functional group (-NH3 +) β2-GP I is complexed to negatively charged phospholipids functional group (-PO4 - ) resulting in the destruction of bilayers and the deactivation of phospholipid molecules.

Dissecting the Acid Stress Response of Rhizobium tropici CIAT 899.

Rhizobium tropici CIAT899 is a nodule-forming α-proteobacterium displaying intrinsic resistance to several abiotic stress conditions such as low pH and high temperatures, which are common in tropical environments. It is a good competitor for Phaseolus vulgaris (common bean) nodule occupancy at low pH values, however little is known about the genetic and physiological basis of the tolerance to acidic conditions. To identify genes in R. tropici involved in pH stress response we combined two different approaches: (1) A Tn5 mutant library of R. tropici CIAT899 was screened and 26 acid-sensitive mutants were identified. For 17 of these mutants, the transposon insertion sites could be identified. (2) We also studied the transcriptomes of cells grown under different pH conditions using RNA-Seq. RNA was extracted from cells grown for several generations in minimal medium at 6.8 or 4.5 (adapted cells). In addition, we acid-shocked cells pre-grown at pH 6.8 for 45 min at pH 4.5. Of the 6,289 protein-coding genes annotated in the genome of R. tropici CIAT 899, 383 were differentially expressed under acidic conditions (pH 4.5) vs. control condition (pH 6.8). Three hundred and fifty one genes were induced and 32 genes were repressed; only 11 genes were induced upon acid shock. The acid stress response of R. tropici CIAT899 is versatile: we found genes encoding response regulators and membrane transporters, enzymes involved in amino acid and carbohydrate metabolism and proton extrusion, in addition to several hypothetical genes. Our findings enhance our understanding of the core genes that are important during the acid stress response in R. tropici.

Influence of Solvent Representation on Nuclear Shielding Calculations of Protonation States of Small Biological Molecules.

In this study, we assess the influence of solvation on the accuracy and reliability of isotropic nuclear magnetic shielding calculations for amino acids in comparison to experimental data. We focus particularly on the performance of solvation methods for different protonation states, as biological molecules occur almost exclusively in aqueous solution and are subject to protonation with pH. We identify significant shortcomings of current implicit solvent models and present a hybrid solvation approach that improves agreement with experimental data by taking into account the presence of direct interactions between amino acid protonation state and water molecules.

Investigation of Non-covalent Interactions of 18-Crown-6 with Amino Acids in Gas Phase by Mass Spectrometry

Abstract The non-covalent interactions between 18-Crown-6 (18c6) and 20 common types of protonated amino acids were explored by electrospray ionization mass spectrometry. The mass spectra showed that 18c6 could react with amino acids to form a non-covalent complexe in a stoichiometric ratio of 1:1. The calibration curves and linear equations for the complexes of L -Phe, L -Tyr, L -Lys and L -Asp with 18c6 were established by mass spectrometric titration and used as reference values for competitive ESI-MS. Through competitive equilibria, the binding constants for the complexes of 18c6 with other L -amino acids and their D -isomers were derived. It was found that, as a general trend, lg K a for the complexes of 18c6 with the basic amino acid and the amino acid with alkyl side chain were larger than other complexes, and among the amino acid with alkyl side chain, Gly and Ala exhibited greater 18c6 binding affinities. As for Ser and Thr, the intramolecular hydrogen bond between the nitrogen atom from terminal –NH 2 and the oxygen atom from carboxyl might impede their protonated amino-group to attack the 18c6. Furthermore, Gln and Asn exhibited lower binding affinities to 18c6, probably due to effects of electron-withdrawing group of acylamide. Finally, the chiral selectivity of 18c6 for L -amino or D -amino acids were measured by ESI-MS, and the result showed that 18c6 could only recognize some neutral amino acid isomers.

Structural and dynamical characterization of the pH-dependence of the pectin methylesterase–pectin methylesterase inhibitor complex

Pectin methylesterases (PMEs) catalyze the demethylesterification of pectin, one of the main polysaccharides in the plant cell wall, and are of critical importance in plant development. PME activity generates highly negatively charged pectin and mutates the physiochemical properties of the plant cell wall such that remodeling of the plant cell can occur. PMEs are therefore tightly regulated by proteinaceous inhibitors (PMEIs), some of which become active upon changes in cellular pH. Nevertheless, a detailed picture of how this pH-dependent inhibition of PME occurs at the molecular level is missing. Herein, using an interdisciplinary approach that included homology modeling, MD simulations, and biophysical and biochemical characterizations, we investigated the molecular basis of PME3 inhibition by PMEI7 in Arabidopsis thaliana Our complementary approach uncovered how changes in the protonation of amino acids at the complex interface shift the network of interacting residues between intermolecular and intramolecular. These shifts ultimately regulate the stability of the PME3-PMEI7 complex and the inhibition of the PME as a function of the pH. These findings suggest a general model of how pH-dependent proteinaceous inhibitors function. Moreover, they enhance our understanding of how PMEs may be regulated by pH and provide new insights into how this regulation may control the physical properties and structure of the plant cell wall.

Assessment of Methodology and Chemical Group Dependences in the Calculation of the pKa for Several Chemical Groups.

We have investigated the dependencies of various computational methods in the calculation of acid dissociation constants (pKa values) of certain chemical groups found in protonatable amino acids based on our previous scheme [ Matsui ; Phys. Chem. Chem. Phys. 2012 , 14 , 4181 - 4187 ]. By changing the quantum chemical (QC) method (Hartree-Fock (HF) and perturbation theory, and composite methods, or exchange-correlation functionals in density functional theory (DFT)), basis sets, solvation models, and the cavities used in the solvent models, we have exhaustively tested about 2,200 combinations to find the best combination for pKa estimation among them. Of the tested parameters, the choice of the basis set and cavity is the most crucial to reproduce experimental values compared to other factors. Concerning the basis set, the inclusion of diffuse functions is quite important for carboxyl, thiol, and phenol groups judging from the mean absolute errors (MAEs) measured from the experimental values. Of the cavity models, between the Pauling, Klamt, and the universal force field (UFF) definitions, the UFF defined cavity is the best choice, resulting in the smallest MAEs. Concerning the QC methods, hybrid DFTs and range-separated DFTs always provide better results than pure DFTs and HF. As a result, we found that LC-ϖPBE/6-31+G(d) with PCM-SMD/UFF provides the best pKa estimation with a MAE within 0.15 pKa units.

Spectroscopic Discrimination of Diastereomeric Complexes Involving an Axially Chiral Receptor.

The infrared multiphoton dissociation (IRMPD) spectra of electrospray ionization (ESI)-formed proton-bound complexes between the axially chiral multifunctional macrocycle MaR and d- and l-phenylalanine (PD and PL ) or d- and l-3,4-dihydroxyphenylalanine (DD and DL ) are recorded in the ν˜ =2800-3700 cm-1 region. Whereas the diastereomeric [MaR ⋅H⋅PD ]+ and [MaR ⋅H⋅PL ]+ complexes do not show any significant spectral differences, the spectrum of [MaR ⋅H⋅DD ]+ clearly diverges from that of its [MaR ⋅H⋅DL ]+ diastereomer. A comparison of the experimental spectra with the B3LYP/6-31G**-calculated harmonic vibrational frequencies of a number of stable structures indicates the formation, in the ESI source, of very similar spectroscopically active structures, with the protonated amino acid placed outside the flat cavity of the macrocycle and hydrogen-bonded at its O2 center. Different spectral signatures of the [MaR ⋅H⋅DD ]+ and [MaR ⋅H⋅DL ]+ complexes are attributed to the coexistence of several stable rotamers in the ESI source.

Side-chain effects on the structures of protonated amino acid dimers: A gas-phase infrared spectroscopy study

A protonated amino acid can interact in several ways with another uncharged amino acid molecule to form a protonated dimer. In case of amino acids that do not have basic or acidic side chains, the most likely protonation site is the amino group and the then protonated amine can be involved in a pairwise interaction with a neutral amine, a carboxylic acid, a carboxylate group and/or the sidechain of the partner amino acid. Here, we employ gas-phase infrared spectroscopy and density functional theory to identify these pairwise interactions in protonated homodimers of serine, isoleucine, phenylalanine and tyrosine. The results show the influence of the different side-chains on the respective interactions. A charge-solvated structure with pairwise interaction between a protonated amine and a neutral amine is preferred if the side chain can provide additional stabilizing interaction with the positive charge. In contrast, for amino acids where the side chain only interacts weakly with the protonated amine group, a protonated dimer is formed by an interaction between the protonated amine and the neutral carboxylic acid of the second amino acid.

Classical Dynamics Simulations of Dissociation of Protonated Tryptophan in the Gas Phase.

Gas phase decomposition of protonated amino acids are of great interest due to their role in understanding protein and peptide chemistry. Several experimental and theoretical studies have been reported in the literature on this subject. In the present work, decomposition of the aromatic amino acid protonated tryptophan was studied by on-the-fly classical chemical dynamics simulations using density functional theory. Mass spectrometry and electronic structure theory studies have shown multiple dissociation pathways for this biologically relevant molecule. Unlike aliphatic amino acids, protonated tryptophan dissociates via NH3 elimination rather than the usual iminium ion formation by combined removal of H2O and CO molecules. Also, a major fragmentation pathway in the present work involves Cα-Cβ bond fission. Results of the chemical dynamics simulations reported here are in overall agreement with experiments, and detailed atomic level mechanisms are presented.

The effect of homocysteine and homocystine protonation on double-layer parameters at the electrode/chlorates(VII) interface

In this study, a comparison of the properties of homocysteine and homocystine adsorption at mercury/chlorate(VII) interface was done. The adsorption of homocysteine and homocystine is in fact the adsorption of mercury(II) cysteine thiolate and mercury(I) cysteine thiolate. The differences in the capacity curves result probably from the formation of mercurous thiolate at the mercury surface. It seems that the changes in Ez and γz values accompanying the change in the amino acid protonation in the basic electrolyte solution, confirm the altered arrangement of the cysteine mercury thiolates on the electrode surface.

A comparative VUV absorption mass-spectroscopy study on protonated peptides of different size.

The ionization of gas-phase protonated peptides and proteins can induce molecular responses ranging from purely non-dissociative ionization to extensive multifragmentation of the system. In the case of soft X-ray photoionization, a monotonic transition between both regimes occurs in the mass range between 0.5 and 10 kDa. Despite the localized nature of the photoabsorption, excitation energy equilibrates before fragmentation sets in and the transition reflects the increase of the heat capacity with protein size. Here, we have investigated the influence of peptide size on vacuum ultraviolet (VUV) photoionization of protonated proteins, where photoexcitation and ionization are limited to valence electrons rather than inner shell electrons and the photoexcitation contribution is markedly lower. Gas phase protonated peptides with masses ranging from 0.6-2.8 kDa were trapped in a radiofrequency ion trap and exposed to synchrotron radiation. Time of flight mass spectrometry was employed for the investigation of the photoionization and photofragmentation processes. The relationship between peptide fragmentation and peptide size exhibits a similar trend as observed for soft X-ray absorption. Due to the lower excitation energies involved, however, dissociation is already quenched at smaller masses and peptide amino acid compositions, protonation states and ionization potentials lead to deviations from the general trend.

Deamidation Reactions of Asparagine- and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy

Deamidation is a major fragmentation channel upon activation by collision induced dissociation (CID) for protonated peptides containing glutamine (Gln) and asparagine (Asn) residues. Here, we investigate these NH3-loss reactions for four Asn- and Gln-containing protonated peptides in terms of the resulting product ion structures using infrared ion spectroscopy with the free electron laser FELIX. The influence of the side chain length (Asn versus Gln) and of the amino acid sequence on the deamidation reaction has been examined. Molecular structures for the product ions are determined by comparison of experimental IR spectra with spectra predicted by density functional theory (DFT). The reaction mechanisms identified for the four dipeptides AlaAsn, AsnAla, AlaGln, and GlnAla are not the same. For all four dipeptides, primary deamidation takes place from the amide side chain (and not from the N-terminus) and, in most cases, resembles the mechanisms previously identified for the protonated amino acids asparagine and glutamine. Secondary fragmentation reactions of the deamidation products have also been characterized and provide further insight in – and confirmation of – the identified mechanisms. Overall, this study provides a comprehensive molecular structure map of the deamidation chemistry of this series of dipeptides. Graphical ᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1462-5) contains supplementary material, which is available to authorized users.

BerlinTrap: A new cryogenic 22-pole ion trap spectrometer

The design and first applications of a new tandem mass spectrometer (BerlinTrap) combining an electrospray ion source, a quadrupole mass spectrometer, a cryogenic 22-pole ion trap (4–300K), and an orthogonal reflectron time-of-flight mass spectrometer are described. The trapped ions are cooled by helium buffer gas cooling. The formation and solvation shell structure of weakly-bound HenH3O+ complexes and the electronic photodissociation spectrum of the protonated amino acid tyrosine are used to calibrate the setup for cooling, tagging, and spectroscopic capabilities. A vibrational temperature below 20K is inferred for protonated tyrosine. The electronic spectrum of isolated protonated lumichrome, the smallest protonated flavin, is recorded in the visible range and assigned to the most stable N5 isomer by comparison with quantum chemical calculations. These results demonstrate the suitability of the BerlinTrap apparatus for spectroscopy and reactivity studies of small and large (bio-)molecular ions and their clusters.

Theoretical study of host–guest interactions in complexes of cucurbit[7]uril with protonated amino acids

The theoretical study of host–guest interactions in complexes of protonated amino acids (AA = Val, Thr, Ser, Asn) and macrocyclic cavitand cucurbit[7]uril with the focus on characterisation of non-covalent interactions between the host and the guest molecules have been carried out. Host–guest interactions have been described by means of R. Bader’s ‘Atoms in Molecules’ theory and Energy Decomposition Analysis (EDA) as it is implemented in ADF program suit. EDA shows that energies of inclusion and exclusion complexes are very close. The optimised geometries for exclusion complexes of CB[7] with protonated AA are slightly more stable than their inclusion counterparts.

Collision Cross Sections for 20 Protonated Amino Acids: Fourier Transform Ion Cyclotron Resonance and Ion Mobility Results.

We report relative dephasing cross sections for the 20 biogenic protonated amino acids measured using the cross sectional areas by Fourier transform ion cyclotron resonance (CRAFTI) technique at 1.9 keV in the laboratory reference frame, as well as momentum transfer cross sections for the same ions computed from Boltzmann-weighted structures determined using molecular mechanics. Cross sections generally increase with increasing molecular weight. Cross sections for aliphatic and aromatic protonated amino acids are larger than the average trend, suggesting these side chains do not fold efficiently. Sulfur-containing protonated amino acids have smaller than average cross sections, reflecting the mass of the S atom. Protonated amino acids that can internally hydrogen-bond have smaller than average cross sections, reflecting more extensive folding. The CRAFTI measurements correlate well with results from drift ion mobility (IMS) and traveling wave ion mobility (TWIMS) spectrometric measurements; CRAFTI results correlate with IMS values approximately as well as IMS and TWIMS values from independent measurements correlate with each other. Both CRAFTI and IMS results correlate well with the computed momentum transfer cross sections, suggesting both techniques provide accurate molecular structural information. Absolute values obtained using the various methods differ significantly; in the case of CRAFTI, this may be due to errors in measurements of collision gas pressure, measurement of excitation voltage, and/or dependence of cross sections on kinetic energy. Graphical Abstract ᅟ.

The effects of homocysteine protonation on double layer parameters at the electrode/chlorates (VII) interface, as well as the kinetics and the mechanism of Bi (III) ion electroreduction

The influence of homocysteine on double layer parameters at the electrode/chlorates (VII) interface and multi-step Bi (III) ion electroreduction was investigated using both voltammetry and impedance methods. The catalytic activity of homocysteine increases with an increasing amount of NaClO4 in a basic electrolyte solution. The highest catalytic activity of homocysteine was observed for 2mol·dm−3 chlorates (VII). The multistage process is confirmed by the not rectilinear lnkf=f(E) dependences. In this mechanism, the chemical stages of Bi–Hg(SR)2 complexes formation with the participation of homocysteine, which is a mediator in the formation of these complexes, and water can be distinguished. The composition of these complexes is diversified in solutions that differ in the degree of amino acid protonation.

Metabolite release and protein hydrolysis during the in vitro digestion of cooked sea bass fillets. A study by 1H NMR

Proton Nuclear Magnetic Resonance (1H NMR) was used to study micro-wave cooked European sea bass (Dicentrarchus labrax) fillets, along five different points of their in vitro digestion: after oral digestion, after gastric digestion and subsequent increasing of the pH to 5.0, and after 90 and 180min of duodenal digestion. During the gastric phase, the appearance of broad signals in different spectral regions, typical of amino acid protons, provided evidence for the expected release of unstructured fragments of denatured proteins, whilst smaller peptides, with sharper signals in the same spectral regions, were becoming visible only after duodenal digestion. Prior digestion, the spectral features of trichloroacetic acid extracts, from raw and cooked fish fillets, have been analysed to assess whether a significant protein hydrolysis or metabolite release (e.g., amino acids, organic acids, saccharides, and nucleotide derivatives) already occurred during cooking, by comparing the spectral profiles of these extracts. No significant differences among the molecular profile of both extracts were found. The present study demonstrates the suitability of NMR spectroscopy to follow the kinetics of fish fillets digestion under in vitro conditions simulating human digestion.

An investigation of folic acid-protein association sites and the effect of this association on folic acid self-assembly.

The contribution of folic acid (FA)-tryptophan interactions to FA-protein association was investigated in the context of using FA as a drug carrier in protein delivery systems. Bovine serum albumin (BSA) and indolicidin were used as model proteins in the study. The FA-BSA complex was characterized by using the Bradford reagent to identify the impact of FA-BSA association on BSA-dye reagent interactions. UV-visible spectroscopic analysis of the FA-BSA mixture showed that the absorbance maximum of BSA-dye reagent occurred at 595nm, even after the association of FA with BSA. This confirms that protonated amino acid groups of the protein are not involved in FA-BSA association. Moreover, molecular dynamics (MD) simulation confirmed the presence of an associative interaction between aromatic moieties in FA and tryptophan moieties in the indolicidin molecule, which disrupted FA self-assembly. An X-ray diffraction (XRD) study showed that there was limited disruption of FA self-assembly after the addition of BSA or tryptophan. This suggests that FA and BSA are compatible and associate with each other. Graphical Abstract Mechanism of folic acid and protein association.

Exploring Strong Interactions in Proteins with Quantum Chemistry and Examples of Their Applications in Drug Design.

ObjectivesThree strong interactions between amino acid side chains (salt bridge, cation-π, and amide bridge) are studied that are stronger than (or comparable to) the common hydrogen bond interactions, and play important roles in protein-protein interactions.MethodsQuantum chemical methods MP2 and CCSD(T) are used in calculations of interaction energies and structural optimizations.ResultsThe energies of three types of amino acid side chain interactions in gaseous phase and in aqueous solutions are calculated using high level quantum chemical methods and basis sets. Typical examples of amino acid salt bridge, cation-π, and amide bridge interactions are analyzed, including the inhibitor design targeting neuraminidase (NA) enzyme of influenza A virus, and the ligand binding interactions in the HCV p7 ion channel. The inhibition mechanism of the M2 proton channel in the influenza A virus is analyzed based on strong amino acid interactions.Conclusion(1) The salt bridge interactions between acidic amino acids (Glu- and Asp-) and alkaline amino acids (Arg+, Lys+ and His+) are the strongest residue-residue interactions. However, this type of interaction may be weakened by solvation effects and broken by lower pH conditions. (2) The cation- interactions between protonated amino acids (Arg+, Lys+ and His+) and aromatic amino acids (Phe, Tyr, Trp and His) are 2.5 to 5-fold stronger than common hydrogen bond interactions and are less affected by the solvation environment. (3) The amide bridge interactions between the two amide-containing amino acids (Asn and Gln) are three times stronger than hydrogen bond interactions, which are less influenced by the pH of the solution. (4) Ten of the twenty natural amino acids are involved in salt bridge, or cation-, or amide bridge interactions that often play important roles in protein-protein, protein-peptide, protein-ligand, and protein-DNA interactions.