Titratable Amino Acid Research Articles

Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of respiratory complex I

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Robust Prediction of Relative Binding Energies for Protein–Protein Complex Mutations Using Free Energy Perturbation Calculations

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein–protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how Protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study.

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Transcendent Aspects of Proton Channels.

A handful of biological proton-selective ion channels exist. Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. This review focuses on common features that result from the unique properties of protons. Proton conduction through water or proteins differs qualitatively from that of all other ions. Extraordinary proton selectivity is needed to ensure that protons permeate and other ions do not. Proton selectivity arises from a proton pathway comprising a hydrogen-bonded chain that typically includes at least one titratable amino acid side chain. The enormously diverse functions of proton channels in disparate regions of the phylogenetic tree can be summarized by considering the chemical and electrical consequences of proton flux across membranes. This review discusses examples of cells in which proton efflux serves to increase pHi, decrease pHo, control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.

Revealing the correlation between the bacterial communities and physicochemical properties of ZaoPoCu, a traditional fermented food from Hainan Island

ABSTRACT The bacterial communities of Zaopocu (ZPC), a tropical specialty fermented food in Hainan Island, were characterized in this study using high-throughput sequencing. Firmicutes and Proteobacteria were the two dominant phyla. The primary genus in ZPC_A was Weissella, while ZPC_B and ZPC_C had Lactobacillus as the dominant genus. The biomarker lactic acid bacteria (LAB) in ZPC_A were Lactobacillus brevis, Weissella confusa, and Leuconostoc citreum, whereas Lactobacillus amylolyticus and Lactobacillus helveticus were significantly higher in ZPC_B. In contrast, Lactobacillus pontis and Lactobacillus fermentum were the core LAB in ZPC_C. ZPC_A has the highest value of titratable acid (TA), amino acid, and lactic acid. TA had the most powerful influence on the structure of the bacterial communities, whereas salinity had a negligible effect. It is worth noting that ZPC may also faces food safety issue because opportunistic pathogenic bacteria like Stenotrophomonas and genes closely associated with Staphylococcus aureus infection were highly detected.

H+ flux of the voltage-gated proton channel Hv1 in giant unilamellar vesicle (GUV).

The voltage-sensitive proton selective channel HV1 is ubiquitously present in mammalian tissues. An aqueous pore provides the proton pathway. Intervening amino acid side chains render the channel impermeable to water [1]. Based on the hydrogen bonds formed between the permeating water molecules and the channel wall, HV1 would otherwise exhibit a water conductance comparable to aquaporins [2,3]. The proton wire involves Grotthuss-like proton hopping on top of intraluminal water molecules and titratable amino acids. Conceivably, HV1 exploits one or more of the arginines from the voltage sensor as part of the proton wire. Here we measured the unitary H+ transport rate by reconstituting the purified proton channel into giant unilamellar vesicles (GUV). We used the pHluorin tag to measure intravesicular pH and oxanol to probe the membrane potential. Fluorescence correlation spectroscopy served to measure channel density. The simultaneous assessment of diffusion limitations of H+ transport towards and away from the channel allows for evaluating the role of HV1's putative proton antenna. This project was supported by the Marie Skłodowska-Curie grant 860592 (Horizon 2020).

Trapped Pore Waters in the Open Proton Channel HV 1.

The voltage-gated proton channel, HV 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3×10-12 cm3 s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the HV 1 wild-type channel and the D174A mutant open at 0mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while interrupting the water wire across the membrane.

Amino acid deprotonation rates from classical force fields.

Acid ionization constants (pKa's) of titratable amino acid side chains have received a large amount of experimental and theoretical attention. In many situations, however, the rates of protonation and deprotonation, kon and koff, may also be important, for example, in understanding the mechanism of action of proton channels or membrane proteins that couple proton transport to other processes. Protonation and deprotonation involve the making and breaking of covalent bonds, which cannot be studied by classical force fields. However, environment effects on the rates should be captured by such methods. Here, we present an approach for estimating deprotonation rates based on Warshel's extension of Marcus's theory of electron transfer, with input from molecular simulations. The missing bond dissociation energy is represented by a constant term determined by fitting the pKa value in solution. The statistics of the energy gap between protonated and deprotonated states is used to compute free energy curves of the two states and, thus, free energy barriers, from which the rate can be deduced. The method is applied to Glu, Asp, and His in bulk solution and select membrane proteins: the M2 proton channel, bacteriorhodopsin, and cytochrome c oxidase.

Prediction of protein pK a with representation learning.

The behavior of proteins is closely related to the protonation states of the residues. Therefore, prediction and measurement of pKa are essential to understand the basic functions of proteins. In this work, we develop a new empirical scheme for protein pKa prediction that is based on deep representation learning. It combines machine learning with atomic environment vector (AEV) and learned quantum mechanical representation from ANI-2x neural network potential (J. Chem. Theory Comput. 2020, 16, 4192). The scheme requires only the coordinate information of a protein as the input and separately estimates the pKa for all five titratable amino acid types. The accuracy of the approach was analyzed with both cross-validation and an external test set of proteins. Obtained results were compared with the widely used empirical approach PROPKA. The new empirical model provides accuracy with MAEs below 0.5 for all amino acid types. It surpasses the accuracy of PROPKA and performs significantly better than the null model. Our model is also sensitive to the local conformational changes and molecular interactions.

Unravelling Constant pH Molecular Dynamics in Oligopeptides with Explicit Solvation Model.

An accurate description of the protonation state of amino acids is essential to correctly simulate the conformational space and the mechanisms of action of proteins or other biochemical systems. The pH and the electrochemical environments are decisive factors to define the effective pKa of amino acids and, therefore, the protonation state. However, they are poorly considered in Molecular Dynamics (MD) simulations. To deal with this problem, constant pH Molecular Dynamics (cpHMD) methods have been developed in recent decades, demonstrating a great ability to consider the effective pKa of amino acids within complex structures. Nonetheless, there are very few studies that assess the effect of these approaches in the conformational sampling. In a previous work of our research group, we detected strengths and weaknesses of the discrete cpHMD method implemented in AMBER when simulating capped tripeptides in implicit solvent. Now, we progressed this assessment by including explicit solvation in these peptides. To analyze more in depth the scope of the reported limitations, we also carried out simulations of oligopeptides with distinct positions of the titratable amino acids. Our study showed that the explicit solvation model does not improve the previously noted weaknesses and, furthermore, the separation of the titratable amino acids in oligopeptides can minimize them, thus providing guidelines to improve the conformational sampling in the cpHMD simulations.

ATR-FTIR Spectroelectrochemical Study on the Mechanism of the pH Dependence of the Redox Potential of the Non-Heme Iron in Photosystem II.

The non-heme iron that bridges the two plastoquinone electron acceptors, QA and QB, in photosystem II (PSII) is known to have a redox potential (Em) of ∼+400 mV with a pH dependence of ∼-60 mV/pH. However, titratable amino acid residues that are coupled to the redox reaction of the non-heme ion and responsible for its pH dependence remain unidentified. In this study, to clarify the mechanism of the pH dependent change of Em(Fe2+/Fe3+), we investigated the protonation structures of amino acid residues correlated with the pH-induced Em(Fe2+/Fe3+) changes using Fourier transform infrared (FTIR) spectroelectrochemistry combined with the attenuated total reflection (ATR) and light-induced difference techniques. Flash-induced Fe2+/Fe3+ ATR-FTIR difference spectra obtained at different electrode potentials in the pH range of 5.0-8.5 showed a linear pH dependence of Em(Fe2+/Fe3+) with a slope of -52 mV/pH close to the theoretical value at 10 °C, the measurement temperature. The spectral features revealed that D1-H215, a ligand to the non-heme iron interacting with QB, was deprotonated to an imidazolate anion at higher pH with a pKa of ∼5.6 in the Fe3+ state, while carboxylate groups from Glu/Asp residues present on the stromal side of PSII were protonated at lower pH with a pKa of ∼5.7 in the Fe2+ state. It is thus concluded that the deprotonation/protonation reactions of D1-H215 and Glu/Asp residues located near the non-heme iron cause the pH-dependent changes in Em(Fe2+/Fe3+) at higher and lower pH regions, respectively, realizing a linear pH dependence over a wide pH range.

Development of a defined autochthonous starter through dissecting the seasonal microbiome of broad bean paste

Bean-based fermentation foods are usually ripened in open environment, which would lead to inconsistencies in flavor and quality between batches. The physicochemical metabolism and microbial community of seasonal broad bean paste (BBP) were compared to distinguish discriminant metabolites and unique taxa, as well as their specific reasons for different flavor and quality in this study. Here, we found that environmental variables led to the seasonal distribution of microbiota, and differential microorganisms further contributed to the inconsistency of flavor quality, in which Lactobacillales was responsible for the higher titratable acid and amino acid nitrogen concentration in winter pei, while Saccharomycetales benefited the formation of volatile flavor substances in autumn pei. Additionally, we compared the effect of different combinations of Lactobacillales with Zygosaccharomyces rouxii on the quality of BBP, and found that W. confusa was more suitable for BBP fermentation rather than T. halophilus in terms of sensory characteristics and physicochemical metabolites.

Fully Atomistic Multiscale Approach for pKa Prediction.

The ionization state of titratable amino acids strongly affects proteins structure and functioning in a large number of biological processes. It is therefore essential to be able to characterize the pKa of ionizable groups inside proteins and to understand its microscopic determinants in order to gain insights into many functional properties of proteins. A big effort has been devoted to the development of theoretical approaches for the prediction of deprotonation free energies, yet the accurate theoretical/computational calculation of pKa values is recognized as a current challenge. A methodology based on a hybrid quantum/classical approach is here proposed for the computation of deprotonation free energies. The method is applied to calculate the pKa of formic acid, methylammonium, and methanethiol, providing results in good agreement with the corresponding experimental estimates. The pKa is also calculated for aspartic acid and lysine as single residues in solution and for three aspartic/glutamic acids inside a well-characterized protein: hen egg white lysozyme. While for small molecules the method is able to deal with multiple protonation states of all titratable groups, this becomes computationally very expensive for proteins. The calculated pKa values for the single amino acids (except for the zwitterionic aspartic acid) and inside the protein display a systematic shift with respect to the experimental values that suggests that the fine balance between hydrophobic and polar interactions might be not accurately reproduced by the usual classical force-fields, thus affecting the computation of deprotonation free energies. The calculated pKa shifts inside the protein are in good agreement with the corresponding experimental ones (within 1 pKa unit), well reproducing the pKa changes due to the protein environment even in the case of large pKa shifts.

Probing the Accuracy of Explicit Solvent Constant pH Molecular Dynamics Simulations for Peptides.

Protonation states of titratable amino acids play a key role in many biomolecular processes. Knowledge of protonatable residue charges at a given pH is essential for a correct understanding of protein catalysis, inter- and intramolecular interactions, substrate binding, and protein dynamics for instance. However, acquiring experimental values for individual amino acid protonation states of complex systems is not straightforward; therefore, several in silico approaches have been developed to tackle this issue. In this work, we assess the accuracy of our previously developed constant pH MD approach by comparing our theoretically obtained pKa values for titratable residues with experimental values from an equivalent NMR study. We selected a set of four pentapeptides, of adequately small size to ensure comprehensive sampling, but concurrently, due to their charge composition, posing a challenge for protonation state calculation. The comparison of the pKa values shows good agreement of the experimental and the theoretical approach with a largest difference of 0.25 pKa units. Further, the corresponding titration curves are in fair agreement, although the shift of the Hill coefficient from a value of 1 was not always reproduced in simulations. The phase space overlap in Cartesian space between trajectories generated in constant pH and standard MD simulations is fair and suggests that our constant pH MD approach reasonably well preserves the dynamics of the system, allowing dynamic protonation MD simulations without introducing structural artifacts.

Effect of Artificial Ageing Using Different Wood Chips on Physico-chemical, Sensory and Antimicrobial Properties of Apple Tea Wine

Abstract: Freshly prepared apple tea wine (a combination of tea extract and apple juice) is having yeasty and dull flavour, which needs to be improved to increase the acceptability of this product. Therefore, an attempt has been made for artificial ageing of apple tea wine using different wood chips to improve its physico-chemical, sensory and antimicrobial attributes. Different types of wood chips (Quercus spp., Bombax spp. and Acacia spp.) were added respectively (2.5 g/L to the freshly prepared apple tea wine) and allowed for ageing in carboys for the six months at the room temperature. The influence of each wood species on physico-chemical, sensory and antimicrobial attributes was tested upto 6 months of storage. Storage intervals significantly affected all the physico-chemical attributes (except total sugars, volatile acidity, and antioxidant activity), whereas, the addition of wood chips affected titratable acidity, ethanol, higher alcohols, total phenols, and amino acid. Cluster analysis of the physico-chemical attributes data revealed the same and showed that storage intervals exerted more effect on the physico-chemical and antimicrobial properties of the apple tea wine rather than the wood chips. The antimicrobial activity of 6 months aged wine was low as compared to the fresh wine. Among all the wood chips, apple tea wine aged with Quercus spp. possesses a significantly higher score (according to desirability) than the wine aged with other wood chips and control. In nutshell, apple tea wine matured with Quercus spp. wood chips for 6 months were the best with improved physico-chemical and sensory attributes.

Tracking Proton Transfer through Titratable Amino Acid Side Chains in Adaptive QM/MM Simulations.

Ubiquitous throughout biological processes, proton transport usually occurs through the Grotthuss shuttling mechanism, where the hydrated proton exists as a charge or structural defect and is propagated quickly through a network of hydrogen bonds with minimal perturbation to the positions of the involved heavy atoms. The rapid reorganization of the bonding network and changing identity of the migrating proton can cause difficulties in molecular dynamics (MD) simulations. Previously, we formulated a proton indicator that tracks the proton as a structural defect for proton exchange between water molecules. In this work, we extend the proton indicator to treat proton transfer between water and amino acid side chains with titratable functional groups. Of particular interest are histidine, glutamate, and arginine, all of which have titratable groups featuring multiple protonation sites. Comparison with the modified center of excess charge (mCEC) suggests that the proton indicator and mCEC are both comparable in approximating the location of the proton. The location of the proton indicator was then used as the center of the QM subsystem in adaptive quantum-mechanical/molecular-mechanical (QM/MM) simulations of proton transport through a model channel along a path consisting of water and titratable amino acid side chains. In the adaptive QM/MM simulations, atoms were reclassified on the fly in a continuous and smooth manner as QM or MM depending on their distances from the proton indicator. Employing a small, mobile QM subsystem, the adaptive QM/MM simulations were found to be much more efficient than the conventional QM/MM simulations with a large QM subsystem that covered the entire pathway for proton relay.

Quality and microbial flora changes of radish paocai during multiple fermentation rounds

Paocai is a popular fermented vegetable side dish or appetizer in China. This study was to investigate the quality and microbial flora changes of paocai during the multiple rounds of fermentation, in order to provide practical suggestions for the industrial production. A total of 4 kinds of lactic acid bacteria were identified based on morphology and calcium dissolution zone size and further 16s RNA identifications. The paocai quality was evaluated on titratable acid, organic acid, amino acid and volatile components content. The results showed that the brine starters after the 4 rounds of fermentation could significantly accelerate the fermentation process from 6 days to only 1–2 days. After 4th round of fermentation, the quality of radish paocai was roughly unified as regard for the microbial flora, organic acid, free amino acid and volatile components content at the maturation point. Therefore, the brine after 4th round of radish paocai fermentation could be used as starters for radish paocai production in industrial scale.

Computational Tools Unravel Putative Sterol Binding Sites in the Lysosomal NPC1 Protein.

Two proteins have been linked as the critical components in the molecular mechanisms involved in the Niemann Pick type C (NPC) disease: NPC1, a 140 kDa polytopic membrane-bound protein, and the smaller (132 residues), water-soluble NPC2 protein. NPC1 is believed to act in tandem with NPC2, transferring cholesterol and other sterols out of the LE/Lys compartments. Mutations in either NPC1 or NPC2 can lead to an accumulation of cholesterol and lipids in the LE/Lys, the primary phenotype of the NPC disease, but approximately 95% of identified disease-causing mutations have been mapped to the membrane-bound NPC1 protein. Here, we investigate the full length, membrane-bound NPC1 protein computationally using a combination of molecular modeling, docking, and molecular dynamics (MD) simulations. An analysis of titratable amino acid side chains, several buried in protein pockets, reveals several nonstandard protonation states for the low-pH scenario (pH 5) that is realized in the lysosome. Together with the location of these buried amino acids, docking studies have identified putative lipid binding domains that are in close proximity to amino acids that, when mutated, are connected to NPC1 loss-of-function. Using energy analyses and MD simulations, we analyze these domains as potential cholesterol binding sites and propose the possibility of multiple sterol binding pockets enabling the intramolecular transport of sterol molecules to the transmembrane domain.

Coarse-grained model of titrating peptides interacting with lipid bilayers.

Molecular-level computer simulations of peptide aggregation, translocation, and protonation at and in biomembranes are impeded by the large time and length scales involved. We present a computationally efficient, coarse-grained, and solvent-free model for the interaction between lipid bilayers and peptides. The model combines an accurate description of mechanical membrane properties with a new granular representation of the dielectric mismatch between lipids and the aqueous phase. All-atom force fields can be easily mapped onto the coarse-grained model, and parameters for coarse-grained monopeptides accurately extrapolate to membrane permeation free energies for the corresponding dipeptides and tripeptides. Acid-base equilibria of titratable amino acid residues are further studied using a constant-pH ensemble, capturing protonation state changes upon membrane translocation. Important differences between histidine, lysine, and arginine are observed, which are in good agreement with experimental observations.

A Genetically Encoded Ratiometric pH Probe: Wavelength Regulation-Inspired Design of pH Indicators.

Mutants of human cellular retinol-binding protein II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH.