Bulky Amino Acids Research Articles

Preventive effects of the novel antimicrobial peptide Nal-P-113 in a rat Periodontitis model by limiting the growth of Porphyromonas gingivalis and modulating IL-1\u03b2 and TNF-\u03b1 production

BackgroundP-113 (AKRHHGYKRKFH-NH2) is a 12-amino-acid histidine-rich peptide derived from histatin 5 that is highly degradable in high salt concentrations and biological fluids such as serum, plasma and saliva. Nal-P-113, a novel antimicrobial peptide whose histidine residues are replaced by the bulky amino acids β-naphthylalanine, causes the antimicrobial peptide to retain its bactericidal activity even in physiological environments. This study evaluated the effect of the novel antimicrobial peptide Nal-P-113 in a rat periodontitis model and the mechanisms of action of Nal-P-113 for suppressing periodontitis.MethodsPeriodontitis was induced in mandibular first molars in rats receiving a ligature and infected with Porphyromonas gingivalis. Animals were randomly divided into six groups: a, P. gingivalis W83 alone; b, P. gingivalis W83 with 6.25 μg/mL of Nal-P-113; c, P. gingivalis W83 with 25 μg/mL of Nal-P-113; d, P. gingivalis W83 with 100 μg/mL of Nal-P-113; e, P. gingivalis W83 with 400 μg/mL of Nal-P-113; and f, control without P. gingivalis W83 or Nal-P-113. Morphometric analysis was used to evaluate alveolar bone loss. Microbiological assessment of the presence of Porphyromonas gingivalis and total bacteria was performed using absolute quantitative real-time PCR and scanning electron microscopy. Gingival tissue was collected for western blot and immunohistochemical assays of IL-1β and TNF-α levels.ResultsAlveolar bone loss was inhibited by 100 μg/mL or 400 μg/mL of Nal-P-113 compared to the control group (P < 0.05). Lower amounts of P. gingivalis and total bacteria were found in groups d and e compared with group a (P < 0.05). A decrease in the levels of IL-1β and TNF-α was detected in group d and group e compared to the control group (P < 0.05). The amount of P. gingivalis was positively correlated with IL-1β and TNF-α expression in periodontal tissue (P < 0.05).ConclusionsNal-P-113 exhibited protective effects on Porphyromonas gingivalis-induced periodontitis in rats by limiting the amount of bacteria and modulating IL-1β and TNF-α production. The use of Nal-P-113 in vivo might serve as a beneficial preventive or therapeutic approach for periodontitis.

The Role of Charge in Protein Targeting Evolution: (Trends in Cell Biology 26, 894-905, 2016).

Due to a formatting error on our behalf, there is a mistake in Table 1Table 1. For Cyanidioschyzon merolae (Rhodophyte), the gene should be CbbX(nuc), the sequence MLGFTTSLQGISLRIRAPAL, and the charge 2.0. In the Refs column, [68] should be [69], [69] should be [70], [80] should be [81], and [82] should be [83]. We note that this has no influence on the remaining manuscript and its conclusions whatsoever, but apologize for any inconvenience this might have caused.Table 1Exemplary Targeting Sequences of Various OrganismsOrganismsGeneN-terminal sequencesChargeTargetingRefsCyanophora.paradoxa (Glaucophyte)PsaLMAFITAIPAVPSTGAIVPAT0.0Plastid[69]Cyanidioschyzon merolae (Rhodophyte)CbbX(nuc)MLGFTTSLQGISLRIRAPAL2.0Plastid[70]Arabidopsis thaliana (Chlorophyte)AtAPX1MTKNYPTVSEDYKKAVEKCR2.0Dual; Mitoch./Plastid[81]Oryza sativa (Chlorophyte)OsAPX6MAVVHRLLRRGLSAASPLPS3.5Mitochondria[81]OsAPX5MAVVHRILRRGLSAASPLPS3.5Mitochondria[81]OsAPX7MAAQRLAALHAAAPSAFSST1.5Plastid[81]OsAPX8MAERIAASLLPAASPSPAPS1.0Plastid[81]Plasmodium falciparum (Alveolata)SyntheticSKINNYSLINKYKINKYTHING4.5Plastid[83]SyntheticITWILLNEVERTARGETPLASTID−2.0ER[83] View Table in HTML Positively charged amino acids are highlighted in blue, negative ones in red and those that can potentially be phosphorylated in green. The bulky aromatic amino acid phenylalanine is underlined.

Recurrent c.G1636A (p.G546S) mutation of COL2A1 in a Chinese family with skeletal dysplasia and different metaphyseal changes: a case report

BackgroundMutations in the COL2A1 gene cause type II collagenopathies characterized by skeletal dysplasia with a wide spectrum of phenotypic severity. Most COL2A1 mutations located in the triple-helical region, and the glycine to bulky amino acid substitutions (e.g., glycine to serine) in the Gly-X-Y repeat were identified frequently. However, the same COL2A1 mutations are associated with different phenotypes and the genotype-phenotype relationship is still poorly understood. Therefore, the studies of more patients about the recurrent mutations in COL2A1 will be needed for further research to provide more comprehensive clinical and genetic data. In this paper, we report a rare recurrent c.G1636A (p.G546S) mutation in COL2A1 associated with different metaphyseal changes in a Chinese family.Case presentationThe proband (III-3) was the second child of the family with skeletal dysplasia. She was 2 years and 3 months old with disproportional short stature, short neck, pectus carinatum, genu varum, bilateral pes planus, and obvious waddling gait. Notably, she displayed severe metaphyseal lesions, especially typical “dappling” and “corner fracture” appearance, whereas no particular metaphyseal involvement was detected in the proband’s mother (II-3) and elder sister (III-2) in the family. We identified a heterozygous mutation (c.1636G > A) in COL2A1 in the three patients, causing the substitution of glycine to serine in codon 546. Although the same mutation has been reported in two previous studies, the phenotypes of the previous patients were different from those of our patients, and the characteristic “dappling” and “corner fracture” metaphyseal abnormalities were not reported previously.ConclusionsIn this study, we identified a c.G1636A (p.G546S) mutation in the COL2A1 associated with different metaphyseal changes, which was never reported in the literature. Our findings revealed a different causative amino acid substitution (glycine to serine) associated with the “dappling” and “corner fracture” metaphyseal abnormalities, and may provide a useful reference for evaluating the phenotypic spectrum and variability of type II collagenopathies.

Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS. This enabled to point out the key role of an isoleucine residue, I1220, from the fatty acid binding site, which could be targeted by mutagenesis. To address this challenge, TALEN (transcription activator-like effector nucleases)-based genome editing technology was applied for the first time to Yarrowia lipolytica and proved to be very efficient for inducing targeted genome modifications. Among the generated FAS mutants, those having a bulky aromatic amino acid residue in place of the native isoleucine at position 1220 led to a significant increase of myristic acid (C14) production compared to parental wild-type KS. Particularly, the best performing mutant, I1220W, accumulates C14 at a level of 11.6% total fatty acids. Overall, this work illustrates how a combination of molecular modeling and genome-editing technology can offer novel opportunities to rationally engineer complex systems for synthetic biology.

Molecular details of the Raptor-binding motif on Arabidopsis S6 kinase

A putative raptor-binding fragment was identified from Arabidopsis S6 kinase 1 (AtS6K1) N-terminal domain in our previous study. Here, we report a further characterization of this fragment, which identified a 12-amino acid core element absolutely required for the interaction. Although the amino acid sequence of the element per se had no significant homology with the canonical consensus of the TOS (TOR-signaling) motif found in the mammalian TOR (target of rapamycin) kinase substrates, its overall sequence composition is similar to that of the TOS motif in that the acidic and non-polar amino acids residues are arranged in alternating fashion and having one or two of the bulky hydrophobic amino acid (F) buried in the interior. Substitution of this bulky residue completely abolished the binding of the fragment to AtRaptor1, as in the case of the mammalian TOS motif. Taken together with its position relative to the catalytic domain of the kinase, which also shows a resemblance with the TOS motif, these results appear to suggest that this core binding element in the N-terminus of AtS6K1 represents a plant version of the TOS motif.

Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues.

Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii.

Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met177, compared with the equivalent residue Leu196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity.

CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.

Understanding the Role of Chain Flexibility in Amyloid Protein Aggregation through Rationally Designed Protein Sequences

The aggregation of amyloid proteins is associated with a myriad of medical conditions including Alzheimer's disease (AD), diabetes, and Parkinson's disease. While attributable to different amyloid proteins, these proteins all share a common feature: a periodic glycine motif (GxxxG). This glycine motif, associated with increased backbone flexibility, is extended in a number of familial mutations that significantly increase severity of AD. A better understanding of the role that chain flexibility plays in protein aggregation will allow for new and innovative protein engineering strategies for nanotech development as well as give insight into therapeutic strategies.In this study, the glycine motif is targeted via either extension, by introduction of additional periodic glycine, or contraction, by replacement of glycine with a bulky or constrained amino acid. Modifications that extend periodic glycine include those that align with familial AD mutations. To examine how these alterations to the glycine repeat motif impact aggregation kinetics, aggregation was monitored via thioflavin-T fluorescence and fit using a novel kinetic equation that accounts for unique features observed at late stages of aggregation. Aggregation products were visualized using transmission electron microscopy to examine morphological features. In addition, aggregates were fractionated by size exclusion chromatography for size analysis via light scattering and morphology analysis via surface hydrophobicity. Results indicate that increased chain flexibility correlates with faster nucleation as well as a larger quantity of small, intermediate aggregates that exhibit reduced fibril morphology with unchanged surface hydrophobicity. Taken together with observations that smaller aggregates are more physiologically active, these results support the hypothesis that increases in protein chain flexibility, including that associated with familial mutations, may contribute to disease progression.Future work will extend studies to other amyloid proteins, such as amylin and chaplin H.

Analysis of crystallographic structures of Ni(ii) complexes of α-amino acid Schiff bases: elucidation of the substituent effect on stereochemical preferences.

In this study, we performed critical analysis of 13 crystallographic structures of various Ni(ii) complexes of amino acid Schiff bases. The major finding of this work is the significance of a parallel displaced type of aromatic interactions between o-amino-benzophenone and Pro N-benzyl rings. The quality of these aromatic interactions was shown to control the steric environment around the amino acid side-chain, rendering variously substituted Ni(ii) complexes of different thermodynamic stabilities. The discovered structural trend holds true for aliphatic, aromatic and sterically bulky amino acids and can be used for the rational design of new and more efficient chiral ligands for general asymmetric synthesis of tailor-made α-amino acids.

Global profiling of SRP interaction with nascent polypeptides.

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Structural and thermodynamic characterisation of L94F mutant of horse cytochrome c

Mammalian mitochondrial cytochromes c (cyts c) has a conserved Leu94 which is replaced by valine/isoleucine in some lower eukaryotes and prokaryotes. It is expected that nature substituted Leu94 with Val/Ile, for they have similar van der Waals volume and hydrophobicity with a difference in side chain branching only. Reports also suggested the presence of phenylalanine at position 94, which leads to questions: (i) How bulky aromatic amino acid residue fitted at position 94 in cyt c family proteins? (ii) What is the effect of L94F mutation on protein stability and folding? Here, we selected horse cyt-c as a model to answer the second question. We generated L94F mutant of horse cytochrome c and subsequently characterised using far-UV, near-UV and Soret circular dichroism, absorbance, intrinsic and extrinsic ANS (8-anilino-1-napthalenesulfonic acid) fluorescence and dynamic light scattering measurements. We observed that this mutation affects the native state and arrests the protein folding at the molten globule state. Thermal stability of L94F mutant is also measured by spectroscopic techniques and differential scanning calorimetry. The midpoint of thermal denaturation of L94F mutant is 17°C less than wild type. Molecular dynamics simulation study also supports our in vitro observation that this mutant has stable backbone conformation.

A Selective and Slowly Reversible Inhibitor of l-Type Amino Acid Transporter 1 (LAT1) Potentiates Antiproliferative Drug Efficacy in Cancer Cells.

The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 μM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.

Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.

Functional Mechanisms of Desensitization in AMPA Receptors

Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory synaptic transmission in the brain. Desensitization of AMPA receptors following glutamate binding is critical for brain function, and involves rearrangement of the ligand binding domains (LBDs). Recently, a couple of full-length structures of desensitized iGluRs were revealed. One of them, the cryo-EM structure of the GluK2 kainate receptor showed that the ligand binding domains (LBDs) adopt a quasi four-fold symmetric arrangement1. Contrastingly, the GluA2 full-length crystal structure with Fluorowillardine bound showed a disrupted, asymmetric orientation of the LBDs and disrupted ATDs2. We reasoned that these LBD arrangements should be detectable by crosslinking experiments during receptor desensitization. We noticed that cysteine mutants at the interface between subunits A and C, which cross-link receptors in partially-active states when desensitization is blocked3, can also trap desensitized receptors. When we exposed receptors to 100 µM glutamate, crosslinks formed that were much more stable than those seen in the absence of desensitization. Consistent with stabilizing a desensitized state, introduction of bulky amino acids at this subunit interface produced a receptor with slow recovery from desensitization. Using a mutation that strongly stabilized the desensitized state (E713T, Y768R), we were able to change the stability of crosslinks or, in one case, block their formation, indicating that there are multiple desensitized arrangements. Homology modelling guided by these functional data suggests that the stable trapping arises from an arrangement distinct from that observed in the partially-active state. Bibliography 1. Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature (2014). 2. Dürr, K. L. et al. Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States. Cell (2014). 3. Lau, A. Y. et al. A Conformational Intermediate in Glutamate Receptor Activation. Neuron (2013).

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels

ABSTRACTVoltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

Dispensability of the [4Fe‐4S] cluster in novel homologues of adenine glycosylase MutY

7,8-Dihydro-8-deoxyguanine (8oG) is one of the most common oxidative lesions in DNA. DNA polymerases misincorporate an adenine across from this lesion. Thus, 8oG is a highly mutagenic lesion responsible for G:C→T:A transversions. MutY is an adenine glycosylase, part of the base excision repair pathway that removes adenines, when mispaired with 8oG or guanine. Its catalytic domain includes a [4Fe-4S] cluster motif coordinated by cysteinyl ligands. When this cluster is absent, MutY activity is depleted and several studies concluded that the [4Fe-4S] cluster motif is an indispensable component for DNA binding, substrate recognition and enzymatic activity. In the present study, we identified 46 MutY homologues that lack the canonical cysteinyl ligands, suggesting an absence of the [4Fe-4S] cluster. A phylogenetic analysis groups these novel MutYs into two different clades. One clade is exclusive of the order Lactobacillales and another clade has a mixed composition of anaerobic and microaerophilic bacteria and species from the protozoan genus Entamoeba. Structural modeling and sequence analysis suggests that the loss of the [4Fe-4S] cluster is compensated by a convergent solution in which bulky amino acids substitute the [4Fe-4S] cluster. We functionally characterized MutYs from Lactobacillus brevis and Entamoeba histolytica as representative members from each clade and found that both enzymes are active adenine glycosylases. Furthermore, chimeric glycosylases, in which the [4Fe-4S] cluster of Escherichia coli MutY is replaced by the corresponding amino acids of LbY and EhY, are also active. Our data indicates that the [4Fe-4S] cluster plays a structural role in MutYs and evidences the existence of alternative functional solutions in nature.

Enhanced stability of Cu(2+)-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the ATCUN motif.

Copper complexes formed by an amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif have attracted attention as metallodrug candidates that cleave DNA or RNA and inactivate enzymes. Although the stability of the Cu(2+)-ATCUN complex under physiologically relevant conditions is a key factor for medical applications, it has remained unclear. Here we prepared a series of ATCUN peptides by inserting various amino acid residues into positions 1 and 2, and investigated the stability of the Cu(2+)-ATCUN complexes in aqueous solution, blood plasma, and living animals. Systematic pH titration showed that the low basicity of the N-terminal amine of the peptide stabilized the Cu(2+)-ATCUN complex in aqueous solution. Interestingly, the stability of (64)Cu-labeled ATCUN complexes in blood plasma was significantly enhanced by the structural bulkiness and hydrophobicity of the amino acid residues at positions 1 and 2. To validate the in vivo stability, six ATCUN motifs (YYH, VVH, NNH, TTH, GGH, and DDH) were conjugated to a tumor-targeting peptide, octreotide (Oct). The stability of the (64)Cu-ATCUN-Oct complexes in blood plasma showed a similar trend to that of the (64)Cu-ATCUN complexes. The (64)Cu-YYH-Oct complex exhibited the highest stability in blood plasma. According to the positron emission tomography and competitive blocking studies of a tumor-bearing mouse model, (64)Cu-YYH-Oct specifically accumulated in tumors, suggesting that the complex was sufficiently stable to reach its target in vivo. The results show that the structural bulkiness and hydrophobicity of the residues at positions 1 and 2 are key parameters for designing metallodrugs on the basis of the Cu(2+)-ATCUN complex.

Positional effects of hydrophobic non-natural amino acid mutagenesis into the surface region of murine dihydrofolate reductase on enzyme properties

The ability to incorporate non-natural amino acids during protein biosynthesis has greatly broadened the amino acid sequence space available for protein engineering. The increasing number of non-natural amino acids provides novel sidechain chemistries, structures, and functionalities for engineered proteins including enzymes. Therefore, understanding how non-natural amino acid incorporation (non-natural amino acid mutagenesis) affects protein function is crucial. This study investigates the positional effects of hydrophobic non-natural amino acid mutagenesis of the enzyme surface on the enzyme structure and function with the ultimate goal of identification of permissive sites for non-natural amino acid mutagenesis. A bulky, hydrophobic non-natural amino acid, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at the solvent-exposed surface of a murine dihydrofolate reductase (mDHFR) enzyme. Incorporation of 2Nal was performed at six solvent-exposed sites (V43, E44, F142, E143, F179, and E180), generating six mDHFR variants. Incorporation of 2Nal into F142 or F179 did not exhibit any substantial change in the secondary structure and catalytic activities, whereas 2Nal incorporation at hydrophilic Glu sites significantly changed the secondary structure and catalytic activities. Such different responses upon 2Nal incorporation at hydrophobic and hydrophilic residues were likely due to the structural changes caused by the hydrophobicity change upon mutation. A non-conservative mutation of V43 with 2Nal significantly reduced the catalytic activities suggesting that a substantial size change of sidechain also causes the structural changes. These results suggest that even hydrophobic, bulky non-natural amino acids can be incorporated at the enzyme surface without compromising the enzymatic activities.

Ultra-rapid polyamidation reaction of optically active aromatic diacid containing methionine moieties with aromatic diamines under microwave irradiation

A novel aromatic dicarboxylic acid monomer, 5-[4-methylthio-2-(9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboximido)butanoylamino]isophthalic acid was synthesized starting from cis-9,10-dihydro-9,10-ethanoanthracene-11,12-dicarboxylic acid anhydride and L-methionine in four steps. A highly effective, very fast microwave method is applied to synthesize optically active and thermally stable polyamides (PA)s under microwave heating for only 3 min. Generally, better yields are obtained under faster and cleaner reactions when compared to those from conventional heating. Therefore, this method could considerably reduce the synthesis time, cost, and energy. The resulting PAs had inherent viscosities in the range of 0.32–0.59 dL/g. All of the these polymers having bulky anthracenic and amino acid functionality in the side chain showed excellent solubility and readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and N,N-dimethylformamide. PAs were thermally stable, with 10 % weight loss recorded at 400 °C and 450 °C under nitrogen atmosphere, and char yields at 800 °C higher than 41 % and glass transition temperature above 168 °C.