Adjacent Residues Research Articles

Search for Highly Divergent Tandem Repeats in Amino Acid Sequences.

We report a Method to Search for Highly Divergent Tandem Repeats (MSHDTR) in protein sequences which considers pairwise correlations between adjacent residues. MSHDTR was compared with some previously developed methods for searching for tandem repeats (TRs) in amino acid sequences, such as T-REKS and XSTREAM, which focus on the identification of TRs with significant sequence similarity, whereas MSHDTR detects repeats that significantly diverged during evolution, accumulating deletions, insertions, and substitutions. The application of MSHDTR to a search of the Swiss-Prot databank revealed over 15 thousand TR-containing amino acid sequences that were difficult to find using the other methods. Among the detected TRs, the most representative were those with consensus lengths of two and seven residues; these TRs were subjected to cluster analysis and the classes of patterns were identified. All TRs detected in this study have been combined into a databank accessible over the WWW.

Comprehensive Assessment of the Relationship Between Site−2 Specificity and Helix α2 in the Erbin PDZ Domain

PDZ domains are key players in signalling pathways. These modular domains generally recognize short linear C-terminal stretches of sequences in proteins that organize the formation of complex multi-component assemblies. The development of new methodologies for the characterization of the molecular principles governing these interactions is critical to fully understand the functional diversity of the family and to elucidate biological functions for family members. Here, we applied an in vitro evolution strategy to explore comprehensively the capacity of PDZ domains for specific recognition of different amino acids at a key position in C-terminal peptide ligands. We constructed a phage-displayed library of the Erbin PDZ domain by randomizing the binding site−2 and adjacent residues, which are all contained in helix α2, and we selected for variants binding to a panel of peptides representing all possible position−2 residues. This approach generated insights into the basis for the common natural class I and II specificities, demonstrated an alternative basis for a rare natural class III specificity for Asp−2, and revealed a novel specificity for Arg−2 that has not been reported in natural PDZ domains. A structure of a PDZ-peptide complex explained the minimum requirement for switching specificity from class I ligands containing Thr/Ser−2 to class II ligands containing hydrophobic residues at position−2. A second structure explained the molecular basis for the specificity for ligands containing Arg−2. Overall, the evolved PDZ variants greatly expand our understanding of site−2 specificities and the variants themselves may prove useful as building blocks for synthetic biology.

Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability.

Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from Talaromyces leycettanus JCM12802 at an optimal temperature of 70 °C. The melting temperature and specific activity of the obtained mutant T316C/G344C were increased by 10 °C and 36.5%, respectively, compared with the wild-type enzyme. The crystal structure of the T316C/G344C mutant showed no formation of a disulfide bond between the introduced cysteines, indicating a different mechanism than the conventional mechanism underlying improved enzyme thermostability. The cysteine substitutions directly formed a new alkyl hydrophobic interaction and caused conformational changes in the side chains of the adjacent residues Asn315 and Thr343, which in turn caused a local reconstruction of hydrogen bonds. This method greatly improved the thermostability of the enzyme without affecting its activity; thus, our findings are of great significance for both theoretical research and practical applications.

The virus-host interface: Molecular interactions of Alphacoronavirus-1 variants from wild and domestic hosts with mammalian aminopeptidase N.

The Alphacoronavirus‐1 species include viruses that infect numerous mammalian species. To better understand the wide host range of these viruses, better knowledge on the molecular determinants of virus–host cell entry mechanisms in wildlife hosts is essential. We investigated Alphacoronavirus‐1 infection in carnivores using long‐term data on Serengeti spotted hyenas (Crocuta crocuta) and molecular analyses guided by the tertiary structure of the viral spike (S) attachment protein's interface with the host receptor aminopeptidase N (APN). We sequenced the complete 3′‐end region of the genome of nine variants from wild African carnivores, plus the APN gene of 15 wild carnivore species. Our results revealed two outbreaks of Alphacoronavirus‐1 infection in spotted hyenas associated with genetically distinct canine coronavirus type II (CCoVII) variants. Within the receptor binding domain (RBD) of the S gene the residues that directly bind to the APN receptor were conserved in all variants studied, even those infecting phylogenetically diverse host taxa. We identified a variable region within RBD located next to a region that directly interacts with the APN receptor. Two residues within this variable region were under positive selection in hyena variants, indicating that both sites were associated with adaptation of CCoVII to spotted hyena APN. Analysis of APN sequences revealed that most residues that interact with the S protein are conserved in wild carnivores, whereas some adjacent residues are highly variable. Of the variable residues, four that are critical for virus–host binding were under positive selection and may modulate the efficiency of virus attachment to carnivore APN.

Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Mechanism of Pyrroloquinoline Quinone-Dependent Hydride Transfer Chemistry from Spectroscopic and High-Resolution X-ray Structural Studies of the Methanol Dehydrogenase from Methylococcus capsulatus (Bath).

The active site of methanol dehydrogenase (MDH) contains a rare disulfide bridge between adjacent cysteine residues. As a vicinal disulfide, the structure is highly strained, suggesting it might work together with the pyrroloquinoline quinone (PQQ) prosthetic group and the Ca2+ ion in the catalytic turnover during methanol (CH3OH) oxidation. We purify MDH from Methylococcus capsulatus (Bath) with the disulfide bridge broken into two thiols. Spectroscopic and high-resolution X-ray crystallographic studies of this form of MDH indicate that the disulfide bridge is redox active. We observe an internal redox process within the holo-MDH that produces a disulfide radical anion concomitant with a companion PQQ radical, as evidenced by an optical absorption at 408 nm and a magnetically dipolar-coupled biradical in the EPR spectrum. These observations are corroborated by electron-density changes between the two cysteine sulfurs of the disulfide bridge as well as between the bound Ca2+ ion and the O5-C5 bond of the PQQ in the high-resolution X-ray structure. On the basis of these findings, we propose a mechanism for the controlled redistribution of the two electrons during hydride transfer from the CH3OH in the alcohol oxidation without formation of the reduced PQQ ethenediol, a biradical mechanism that allows for possible recovery of the hydride for transfer to an external NAD+ oxidant in the regeneration of the PQQ cofactor for multiple catalytic turnovers. In support of this mechanism, a steady-state level of the disulfide radical anion is observed during turnover of the MDH in the presence of CH3OH and NAD+.

Copper (II) binding properties of an octapeptide fragment from the R3 region of tau protein: A combined potentiometric, spectroscopic and mass spectrometric study

The copper(II) complexes of a peptide fragment of the R3 domain of tau protein (tau(326–333) Ac-GNIHHKPG-NH2) and its mutants (Ac-GNGHHKPG-NH2, Ac-GNIHHKAG-NH2, Ac-GNGAHKPG-NH2 and Ac-GNGHAKPG-NH2) have been studied by potentiometric and spectroscopic (UV–Vis, CD) methods. ESR spectroscopy and mass spectrometry were also used to prove the coordination mode of the mononuclear complexes and the formation of dinuclear species, respectively. It has been demonstrated that the (326–333) fragment of tau protein is a versatile and effective ligand for copper(II) coordination. The versatility of copper(II) binding is related to the presence of two adjacent histidyl residues in the sequence, which results in the coexistence of mononuclear, bis(ligand) and dinuclear complexes at different metal to ligand ratios. The 1:1 mononuclear complexes are, however, the dominant species with all peptides and the imidazole-N and one to three deprotonated amide nitrogen atoms towards the N-terminal side of the histidyl residue have been suggested as metal binding sites. This binding mode allows the formation of coordination isomers because any of the two histidine moieties can be the primary anchoring site. It is evident from the CD spectroscopic measurements that the isomers are present in almost equal concentration. The copper(II) binding affinity of the native fragment of tau protein is comparable to that of a similar 2-histidine fragment of amyloid-β mutant, Ac-SGAEGHHQK-NH2 but the comparison with an independent histidyl residue (H32) from the N-terminal region of the protein reveals the predominance of H32 over the histidines in the R3 domain.

Sequence-dependent nanomolar binding of tripeptides containing N-terminal phenylalanine by Cucurbit[7]uril: A theoretical study

Molecular recognition towards peptides with high affinity and sequence selectivity by synthetic macrocyclic receptors is a great challenge in supramolecular chemistry. In this work, we investigate the molecular recognition of tripeptides containing N-terminal phenylalanine (Phe, F) by synthetic macrocyclic receptor Cucurbit[7]uril (CB[7]) in aqueous solution by molecular dynamics simulations. We found that among twenty amino acids (AAs), the binding of CB[7] to Phe is the strongest. Based on that a series of tripeptides containing N-terminal Phe (with the second residue Gly (G) fixed) are designed to explore the effects of adjacent AA residues on the binding to CB[7]. It is indicated that, in contrast to FGG, only when the 3rd-residue is Glu (E), Lys (K), or Arg (R), the binding affinity with CB[7] can reach the nanomolar level. For the most prominent FGE, the binding free energy with CB[7] is −12.8 kcal/mol, with association constant (Ka) of 2.1 × 109 M−1. It is surprising that once reversing the sequence order of the 2nd-residue and 3rd-residue from FGX to FXG (X = E, K, R), or inserting a second Gly in the middle of FGX, such as FGGX, the binding affinity is significantly changed, with Ka decreased more than 3 orders of magnitude. These results predicted the high binding affinity and sequence selectivity of CB[7] to containing N-terminal Phe peptides, which are beneficial to predict the recognition sites of specific proteins/peptides and to design host-guest complexes with high affinity.

Investigation of action pattern of a novel chondroitin sulfate/dermatan sulfate 4-O-endosulfatase.

Recently, a novel CS/DS 4-O-endosulfatase was identified from a marine bacterium and its catalytic mechanism was investigated further (Wang, W., et. al (2015) J. Biol. Chem.290, 7823-7832; Wang, S., et. al (2019) Front. Microbiol.10, 1309). In the study herein, we provide new insight about the structural characteristics of the substrate which determine the activity of this enzyme. The substrate specificities of the 4-O-endosulfatase were probed by using libraries of structure-defined CS/DS oligosaccharides issued from synthetic and enzymatic sources. We found that this 4-O-endosulfatase effectively remove the 4-O-sulfate of disaccharide sequences GlcUAβ1-3GalNAc(4S) or GlcUAβ1-3GalNAc(4S,6S) in all tested hexasaccharides. The sulfated GalNac residue is resistant to the enzyme when adjacent uronic residues are sulfated as shown by the lack of enzymatic desulfation of GlcUAβ1-3GalNAc(4S) connected to a disaccharide GlcUA(2S)β1-3GalNAc(6S) in an octasaccharide. The 3-O-sulfation of GlcUA was also shown to hinder the action of this enzyme. The 4-O-endosulfatase exhibited an oriented action from the reducing to the non-reducing whatever the saturation or not of the non-reducing end. Finally, the activity of the 4-O-endosulfatase decreases with the increase in substrate size. With the deeper understanding of this novel 4-O-endosulfatase, such chondroitin sulfate (CS)/dermatan sulfate (DS) sulfatase is a useful tool for exploring the structure-function relationship of CS/DS.

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor.

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

Computational Prediction of Intrinsically Disordered Proteins Based on Protein Sequences and Convolutional Neural Networks.

Intrinsically disordered proteins (IDPs) possess at least one region that lacks a single stable structure in vivo, which makes them play an important role in a variety of biological functions. We propose a prediction method for IDPs based on convolutional neural networks (CNNs) and feature selection. The combination of sequence and evolutionary properties is used to describe the differences between disordered and ordered regions. Especially, to highlight the correlation between the target residue and adjacent residues, multiple windows are selected to preprocess the protein sequence through the selected properties. The shorter windows reflect the characteristics of the central residue, and the longer windows reflect the characteristics of the surroundings around the central residue. Moreover, to highlight the specificity of sequence and evolutionary properties, they are preprocessed, respectively. After that, the preprocessed properties are combined into feature matrices as the input of the constructed CNN. Our method is training as well as testing based on the DisProt database. The simulation results show that the proposed method can predict IDPs effectively, and the performance is competitive in comparison with IsUnstruct and ESpritz.

A conserved residue in the P2X4 receptor has a nonconserved function in ATP recognition

Highly conserved amino acids are generally anticipated to have similar functions across a protein superfamily, including that of the P2X ion channels, which are gated by extracellular ATP. However, whether and how these functions are conserved becomes less clear when neighboring amino acids are not conserved. Here, we investigate one such case, focused on the highly conserved residue from P2X4, E118 (rat P2X4 numbering, rP2X4), a P2X subtype associated with human neuropathic pain. When we compared the crystal structures of P2X4 with those of other P2X subtypes, including P2X3, P2X7, and AmP2X, we observed a slightly altered side-chain orientation of E118. We used protein chimeras, double-mutant cycle analysis, and molecular modeling to reveal that E118 forms specific contacts with amino acids in the “beak” region, which facilitates ATP binding to rP2X4. These contacts are not present in other subtypes because of sequence variance in the beak region, resulting in decoupling of this conserved residue from ATP recognition and/or channel gating of P2X receptors. Our study provides an example of a conserved residue with a specific role in functional proteins enabled by adjacent nonconserved residues. The unique role established by the E118-beak region contact provides a blueprint for the development of subtype-specific inhibitors of P2X4.

Targeting mechanism for SARS-CoV-2 in silico: interaction and key groups of TMPRSS2 toward four potential drugs.

The global dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously endangered human health. The number of confirmed cases is still increasing; however, treatment options are limited. Transmembrane protease serine 2 (TMPRSS2), as a key protease that primes the binding of SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2), has become an attractive target and received widespread attention. Thus, four potential drugs (bromhexine, camostat, gabexate, and nafamostat) were used to explore the mechanism of binding with TMPRSS2 in this work. A 65 ns molecular dynamics simulation was performed three times for each drug-TMPRSS2 system for reliable energy calculation and conformational analysis, of which the simulations of nafamostat-TMPRSS2 systems were further extended to 150 ns three times due to the discovery of two binding modes. Through the results of calculating binding free energy by nine methods, the binding affinity of camostat, gabexate, and nafamostat to TMPRSS2 showed great advantages compared with bromhexine, where the nafamostat was surprisingly found to present two reasonable binding conformations (forward and reverse directions). Two negatively charged amino acids (Asp435 and Glu299) can clamp the two positively charged groups (guanidinium group and amidinium group) in either forward or reverse fashion, and the forward one is more stable than the reverse. In addition, compared with gabexate, the dimethylamino group in camostat forms more van der Waals interactions with surrounding hot-spots His296 and Val280, resulting in a stronger affinity to TMPRSS2. For bromhexine, multiple binding sites are displayed in the binding pocket due to its small molecular structure, and van der Waals interactions play the dominant role in the binding process. In particular, six typical hot-spots were identified in the last three serine protease inhibitor systems, i.e., Asp435, Ser436, Gln438, Trp461, Ser463, and Gly464. The guanidinium groups of the drugs have powerful interactions with adjacent residues due to the formation of more hydrogen bonds, suggesting that this may be the critical site for drug design against TMPRSS2. This work provides valuable molecular insight into these four drug-TMPRSS2 binding mechanisms and is helpful for designing and screening drugs targeting TMPRSS2.

Effects of substrate modifications on the arginine dimethylation activities of PRMT1 and PRMT5

ABSTRACT Histone arginine methylation is a prevalent posttranslational modification (PTM) in eukaryotic cells and contributes to the histone codes for epigenetic regulation of gene transcription. In this study, we determined how local changes on adjacent residues in the histone H4 substrate regulate arginine asymmetric dimethylation and symmetric dimethylation catalysed by the major protein arginine methyltransferase (PRMT) enzymes PRMT1 and PRMT5, respectively. We found that phosphorylation at histone H4 Ser-1 site (H4S1) was inhibitory to activities of PRMT1 and PRMT5 in both monomethylating and dimethylating H4R3. Also, a positively charged H4K5 was important for PRMT1 catalysis because acetylation of H4K5 or the loss of the H4K5 ε-amine had a similar effect in reducing the catalytic efficiency of asymmetric dimethylation of H4R3. An opposite effect was observed in that acetylation of H4K5 or the loss of the H4K5 ε-amine enhanced PRMT5-mediated symmetric dimethylation of H4R3. Furthermore, we observed that N-terminal acetylation of H4 modestly decreased asymmetric dimethylation of H4R3 by PRMT1 and symmetric dimethylation of H4R3 by PRMT5. This work highlights the significance of local chemical changes in the substrate to regulating PRMT activity and unravels the pattern complexities and subtleties of histone codes.

An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons.

Neuroligins (NLGNs) are a class of postsynaptic cell adhesion molecules that interact with presynaptic neurexins (NRXNs) and regulate synapse function. NLGN4 is a member of the NLGN family and consists of a unique amino acid sequence in humans that is not evolutionarily well conserved in rodents. The human-specific NLGN4 gene has been reported to be mutated in many patients with autism and other neurodevelopmental disorders. However, it remained unclear how these mutations might alter the molecular properties of NLGN4 and affect synaptic transmission in human neurons. Here, we describe a severely autistic male patient carrying a single amino acid substitution (R101Q) in the NLGN4 gene. When expressed in HEK293 cells, the R101Q mutation in NLGN4 did not affect its binding affinity for NRXNs or its capacity to form homodimers. This mutation, however, impaired the maturation of NLGN4 protein by inhibiting N-linked glycosylation at an adjacent residue (N102), which is conserved in all NLGNs. As a result, the R101Q substitution significantly decreased the surface trafficking of NLGN4 and increased its retention in the endoplasmic reticulum and Golgi apparatus. In human neurons derived from male stem cell lines, the R101Q mutation also similarly reduced the synaptic localization of NLGN4, resulting in a loss-of-function phenotype. This mutation-induced trafficking defect substantially diminished the ability of NLGN4 to form excitatory synapses and modulate their functional properties. Viewed together, our findings suggest that the R101Q mutation is pathogenic for NLGN4 and can lead to synaptic dysfunction in autism.

Conjecture on the Design of Helical Proteins.

In an important advance in our understanding of protein folding, Wolynes and Onuchic found that the frustration ratio, Tf/Ts, for funneled energy landscapes is Tf/Ts ∼1.6. In our recent work on four heme proteins, we showed that when a protein unfolds from the native state to an early unfolded state, the degree of departure is characterized by a ratio f ∼1.6, where f is a measure of the elongation of n-residue segments of the polypeptide chain. Our analysis, which accounts for this apparent similarity in calculated signatures, is based on a logistic-map model of unfolding. We offer an important take home for the de novo protein synthesis community: in order to increase the probability of obtaining good quality crystals, nearest-neighbor repulsive interactions between adjacent residues (or sequences of residues) in the polypeptide chain must be propagated correctly.

Phi class glutathione transferases as molecular targets towards multiple-herbicide resistance: Inhibition analysis and pharmacophore design

Multiple-herbicide resistance (MHR) is a global threat to weed control in cereal crops. MHR weeds express a specific phi class glutathione transferase (MHR-GSTF) that confers resistance against multiple herbicides and therefore represents a promising target against MHR weeds. Kinetics inhibition analysis of MHR-GSTFs from grass weeds Lolium rigidum (LrGSTF) Alopecurus myosuroides (AmGSTF) and crops Hordeum vulgare (HvGSTF) and Triticum aestivum (TaGSTF) allowed the identification of the acetanilide herbicide butachlor as a potent and selective inhibitor towards MHR-GSTFs. Also, butachlor is a stronger inhibitor for LrGSTF and AmGSTF compared to HvGSTF and TaGSTF from crops. The crystal structure of LrGSTF was determined at 1.90 Å resolution in complex with the inhibitor S-(4-nitrobenzyl)glutathione. A specific 3D pharmacophore targeting the MHR-GSTFs was designed and used to identify structural elements important for potent and selective inhibition. Structural analysis of GSTFs revealed a decisive role of conserved Tyr118 in ligand binding and pharmacophore design. Its positioning is dependent on an outer patch of adjacent residues that span from position 132 to 134 which are similar for both LrGSTF and AmGSTF but different in HvGSTF and TaGSTF. The results presented here provide new knowledge that may be adopted to cope with MHR weeds.

Synthesis and bio-evaluation of a novel selective butyrylcholinesterase inhibitor discovered through structure-based virtual screening

In recent years, butyrylcholinesterase (BChE) has gradually gained worldwide interests as a novel target for treating Alzheimer's disease (AD). Here, two pharmacophore models were generated using Schrödinger suite and used to virtually screen ChemDiv database, from which three hits were obtained. Among them, 2513–4169 displayed the highest inhibitory activity and selectivity against BChE (eeAChE IC50 > 10 μM, eqBChE IC50 = 3.73 ± 1.90 μM). Molecular dynamic (MD) simulation validated the binding pattern of 2513–4169 in BChE, and it could form a various of receptor-ligand interactions with adjacent residues. In vitro cytotoxicity assay proved the safety of 2513–4169 on diverse neural cell lines. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay performed on SH-SY5Y cells proved the neuroprotective effect of 2513–4169 against toxic Aβ1–42. In vivo behavioral study further confirmed the great efficacy of 2513–4169 on reversing Aβ1–42-induced cognitive impairment of mice and clearing the toxic Aβ1–42 in brains. Moreover, 2513–4169 was proved to be able to cross blood-brain barrier (BBB) through a parallel artificial membrane permeation assay of BBB (PAMPA-BBB). Taken together, 2513–4169 is a promising lead compound for future optimization to discover anti-AD treating agents.

Quantification of bovine α-lactalbumin in infant milk formula using LC-MS

α-Lactalbumin (α-la) provides essential amino acids to the diet and is therefore of nutritional importance. Bovine α-la is routinely supplemented to infant formulas, which makes it essential to obtain quantitative data of its content. In this study a quantitative, internally standardised Liquid Chromatography-Mass Spectrometry (LC-MS) method for bovine α-la in infant formula was validated, with good accuracy (90–110%) and precision (RSDRint < 7%). The specificity was high because the bovine α-la calibration samples were prepared with goat milk-based infant formula to avoid background of the endogenous bovine target peptide (CEVFR). Additionally, the method includes the qualitative monitoring of (i) a second confirmative tryptic peptide from the bovine α-la sequence (VGINYWLAHK), (ii) glycation of adjacent lysine residues and (iii) the presence of goat α-la adulteration in bovine infant milk formula. The methodology will be of importance to the dairy and infant formula industry in supporting bovine α-la label claims.

Proton transfer pathway from the oxygen-evolving complex in photosystem II substantiated by extensive mutagenesis

We report a structure-based biological approach to identify the proton-transfer pathway in photosystem II. First, molecular dynamics (MD) simulations were conducted to analyze the H-bond network that may serve as a Grotthuss-like proton conduit. MD simulations show that D1-Asp61, the H-bond acceptor of H2O at the Mn4CaO5 cluster (W1), forms an H-bond via one water molecule with D1-Glu65 but not with D2-Glu312. Then, D1-Asp61, D1-Glu65, D2-Glu312, and the adjacent residues, D1-Arg334, D2-Glu302, and D2-Glu323, were thoroughly mutated to the other 19 residues, i.e., 114 Chlamydomonas chloroplast mutant cells were generated. Mutation of D1-Asp61 was most crucial. Only the D61E and D61C cells grew photoautotrophically and exhibit O2-evolving activity. Mutations of D2-Glu312 were less crucial to photosynthetic growth than mutations of D1-Glu65. Quantum mechanical/molecular mechanical calculations indicated that in the PSII crystal structure, the proton is predominantly localized at D1-Glu65 along the H-bond with D2-Glu312, i.e., pKa(D1-Glu65) > pKa(D2-Glu312). The potential-energy profile shows that the release of the proton from D1-Glu65 leads to the formation of the two short H-bonds between D1-Asp61 and D1-Glu65, which facilitates downhill proton transfer along the Grotthuss-like proton conduit in the S2 to S3 transition. It seems possible that D1-Glu65 is involved in the dominant pathway that proceeds from W1 via D1-Asp61 toward the thylakoid lumen, whereas D2-Glu312 and D1-Arg334 may be involved in alternative pathways in some mutants.