Understanding the Enzyme (S)-Norcoclaurine Synthase Promiscuity to Aldehydes and Ketones.

The (S)-norcoclaurine synthase from Thalictrum flavum (TfNCS) stereoselectively catalyzes the Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. TfNCS can catalyze the Pictet-Spengler reaction with various aldehydes and ketones, leading to diverse tetrahydroisoquinolines. This substrate promiscuity positions TfNCS as a highly promising enzyme for synthesizing fine chemicals. Understanding carbonyl-containing substrates' structural and electronic signatures that influence TfNCS activity can help expand its applications in the synthesis of different compounds and aid in protein optimization strategies. In this study, we investigated the influence of the molecular properties of aldehydes and ketones on their reactivity in the TfNCS-catalyzed Pictet-Spengler reaction. Initially, we compiled a library of reactive and unreactive compounds from previous publications. We also performed enzymatic assays using nuclear magnetic resonance to identify some reactive and unreactive carbonyl compounds, which were then included in the library. Subsequently, we employed QSAR and DFT calculations to establish correlations between substrate-candidate structures and reactivity. Our findings highlight correlations of structural and stereoelectronic features, including the electrophilicity of the carbonyl group, to the reactivity of aldehydes and ketones toward the TfNCS-catalyzed Pictet-Spengler reaction. Interestingly, experimental data of seven compounds out of fifty-three did not correlate with the electrophilicity of the carbonyl group. For these seven compounds, we identified unfavorable interactions between them and the TfNCS. Our results demonstrate the applications of in silico techniques in understanding enzyme promiscuity and specificity, with a particular emphasis on machine learning methodologies, DFT electronic structure calculations, and molecular dynamic (MD) simulations.

Chronic alcohol and nicotine consumption as catalyst for systemic inflammatory storm and bone destruction in apical periodontitis.

AIM: To assess the periapical alveolar bone pattern and the serum levels of proinflammatory cytokines, biochemical markers and metabolites in rats subjected to chronic alcohol and nicotine consumption and induced apical periodontitis. METHODOLOGY: Twenty-eight male Wistar rats were divided into four groups: Control, Alcohol, Nicotine and Alcohol+Nicotine. The alcohol groups were exposed to self-administration of a 25% alcohol solution, while the other groups were given only filtered water. The nicotine groups received daily intraperitoneal injections of a nicotine solution (0.19 µL of nicotine/mL), whereas the other groups received saline solution. Periapical lesions were induced by exposing the pulps of the left mandibular first molars for 28 days. After euthanasia, the mandibles were removed and the percentage bone volume, bone mineral density, trabecular thickness, trabecular separation and trabecular number of the periapical bone were measured using micro-computed tomography images. Serum samples were collected for analysis of proinflammatory cytokines (IL-1ß, IL-4, IL-6 and TNF-α), biochemical and metabolomic analysis. Statistical analysis was performed with a significance level of 5%. Nonparametric data were analysed using the Kruskal-Wallis test followed by Dunn's test, while one-way anova followed by Tukey's test was performed for parametric data. RESULTS: The groups exposed to alcohol or nicotine consumption exhibited an altered bone pattern indicating lower bone density and higher levels of IL-1ß, IL-6 and TNF-α compared to the Control group (p < .05). Significant differences were observed among the groups in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, cholesterol, triglycerides, urea, creatinine, albumin, uric acid, bilirubin and calcium. Metabolomic analysis revealed significant differences in glycine, phosphocholine, lysine, lactate, valine, pyruvate and lipids (CH2 CH2 CO), n(CH2 ) and n(CH3 ). Most of these parameters were even more altered in the simultaneous consumption of both substances compared to single consumption. CONCLUSION: Alcohol and nicotine chronic consumption altered several metabolic markers, impaired liver and kidney function, increased the production of systemic proinflammatory mediators and harmed the periapical bone microarchitecture in the presence of apical periodontitis. The simultaneous consumption of alcohol and nicotine intensified these detrimental effects.

Untargeted stimulated and unstimulated salivary metabolomics and saliva flow rate in children.

OBJECTIVE: The present study aimed to determine the salivary flow and metabolomic profile of stimulated and unstimulated saliva in children. MATERIALS AND METHODS: Children who attended the Pediatric Dentistry Clinic of the State University of Rio de Janeiro -UERJ between 3 and 12 years of age were selected. Unstimulated and stimulated whole saliva, using mechanical stimulus, were collected. The samples were centrifuged at 12,000 g, 4oC for 1 h. The 1H- NMR spectra were acquired in 500 MHz equipment. The data were extracted into 0.03 ppm buckets in AMIX, and multivariate analysis (PLS-DA and O-PLS-DA) was performed in Metaboanalyst 2.0. For other analyses, such as salivary flow, the data was tabulated in the SPSS 20.0 statistical package, analyzed descriptively, and after applying the Wilcoxon test. The interval of confidence was set at 95%. RESULTS: The mean age was 7.5 (± 1.94), and 47.0% (n = 31) were female, 63.6% (n = 42). The median flow rate for stimulated saliva was 0.74 (IC 0.10-2.40) and was statistically higher (p < 0.001; Wilcoxon test) than unstimulated was 0.39 (IC 0.00-1.80). Children older than seven years old also presented a higher difference between unstimulated and stimulated saliva (p = 0.003; Mann-Whitney test). The PLS-DA and O-PLS-DA demonstrated a different profile in stimulated and unstimulated saliva. Acetate, glucose, propionate, and lysine were higher in the unstimulated whole saliva than in stimulated saliva. Isoleucine, N-acetyl sugar, hydroxybutyrate, glutamate, leucine, propionate, butyrate, valine, isoleucine, succinate, saturated fatty acid, and histidine were found in greater amounts in the saliva of patients with stimulated saliva. CONCLUSION: The stimulated saliva presented a higher flow rate, and older children exhibited a higher flow rate resulting from it's the stimulus. The mechanical stimulus increased the levels of the major metabolites.

Unjustified use of amber necklaces for teething symptoms alleviation: Succinic acid release underperforms compared with natural skin bacteria production.

BACKGROUND: There is limited evidence of succinic acid release from amber necklace that justifies its biological plausibility. AIM: This study aimed to evaluate the release of succinic acid from Baltic amber beads in the presence of Staphylococcus epidermidis. DESIGN: The Baltic amber beads from the necklace were stratified according to their weight (average 0.05 g ± 0.067). Subsequently, the beads (n = 8) were submerged in 0.9% buffered saline (Control) or brain-heart infusion culture medium in the presence of a commercial strain of S. epidermidis, a resident skin bacterium incubated at 37°C for 24 h or 7 days. The samples were centrifuged, and the supernatants were analyzed by 1H Nuclear Magnetic Resonance. Multivariate analyses were adopted using the sparse partial least squares discriminant analysis method (p < .05). RESULTS: The group incubated with saline solution showed small release of succinic acid only after 7 days. In the groups with S. epidermidis, the release of succinic acid was observed in the both presence and absence of amber beads, indicating that succinic acid is a product released by bacteria. CONCLUSIONS: It was found that amber beads do not exhibit the ability to release expressive succinic acid, especially in a short period of time, which does not justify their use in infants. The most production of succinic acid is tributed to S. epidermidis.

Metabolic Adaptations Correlated with Antibody Response after Immunization with Inactivated SARS-CoV-2 in Brazilian Subjects.

The adsorbed vaccine SARS-CoV-2 (inactivated) produced by Sinovac (SV) was the first vaccine against COVID-19 to be used in Brazil. To understand the metabolic effects of SV in Brazilian subjects, NMR-based metabolomics was used, and the immune response was studied in Brazilian subjects. Forty adults without (group-, n = 23) and with previous COVID-19 infection (group+, n = 17) were followed-up for 90 days postcompletion of the vaccine regimen. After 90 days, our results showed that subjects had increased levels of lipoproteins, lipids, and N-acetylation of glycoproteins (NAG) as well as decreased levels of amino acids, lactate, citrate, and 3-hydroxypropionate. NAG and threonine were the highest correlated metabolites with N and S proteins, and neutralizing Ab levels. This study sheds light on the immunometabolism associated with the use of SV in Brazilian subjects from Rio de Janeiro and identifies potential metabolic markers associated with the immune status.

NMR structure determination of Ixolaris and factor X(a) interaction reveals a noncanonical mechanism of Kunitz inhibition.

Ixolaris is a potent tick salivary anticoagulant that binds coagulation factor Xa (FXa) and zymogen FX, with formation of a quaternary tissue factor (TF)/FVIIa/ FX(a)/Ixolaris inhibitory complex. Ixolaris blocks TF-induced coagulation and PAR2 signaling and prevents thrombosis, tumor growth, and immune activation. We present a high-resolution structure and dynamics of Ixolaris and describe the structural basis for recognition of FX. Ixolaris consists of 2 Kunitz domains (K1 and K2) in which K2 is strikingly dynamic and encompasses several residues involved in FX binding. This indicates that the backbone plasticity of K2 is critical for Ixolaris biological activity. Notably, a nuclear magnetic resonance-derived model reveals a mechanism for an electrostatically guided, high-affinity interaction between Ixolaris and FX heparin-binding (pro)exosite, resulting in an allosteric switch in the catalytic site. This is the first report revealing the structure-function relationship of an anticoagulant targeting a zymogen serving as a scaffold for TF inhibition.

Salivary metabolome of children and adolescents under peritoneal dialysis.

OBJECTIVE: To study the influence of peritoneal dialysis (PD) on the salivary metabolite profile of children and adolescents with renal failure. MATERIALS AND METHODS: Healthy children/adolescents (n = 31; mean age: 12.18 ± 3.76) and children/adolescents subjected to PD (n = 12; mean age: 10.10 ± 4.25) were recruited. Oral health status assessed by the dmft/DMFT and Volpe-Manhold calculus indices. The 1H spectra were acquired in a 600-MHz Bruker nuclear magnetic resonance spectrometer and were subjected to multivariate analysis using partial least squares discriminant analysis (PLS-DA), orthogonal PLS-DA (O-PLS-DA), and univariate analysis through chi-square and t tests (SPSS 20.0, IL, USA), with a significance level of p < 0.05. RESULTS: A similar caries pattern (p = 0.57; chi-square test) was observed between the healthy (dmft = 0.72 ± 1.28 and DMFT 0.93 ± 2.30) and PD groups (dmft = 2.14 ± 3.67, DMFT 0.33 ± 0.71) and dental calculus (p > 0.05, t test). PLS-DA and O-PLS-DA were able to distinguish both groups (ACC = 0.85, R2 = 0.80, Q2 = 0.15). Salivary metabolites decrease in creatine, propionate, and sugar levels in the PD group and an increase in creatinine, butyrate, and lactate levels when compared with the healthy group. CONCLUSIONS: Children and adolescents subjected to PD have a different salivary metabolic profile from that of their healthy subjects. CLINICAL RELEVANCE: Complications of peritoneal dialysis procedures could be monitored by proper knowledge of saliva characteristics as predictors of peritonitis-related outcome. The use of metabolomics in pediatric nephrology may be an innovative methodology for the early diagnosis and monitoring of kidney diseases.

Cytocompatibility of filling pastes by primary teeth root simulating model.

Evaluate the cytocompatibility of Calen®/ZO, Calcicur®, Vitapex®, Endoflas®, and zinc oxide/eugenol-based (ZOE) root canal pastes (RCP) to human primary osteoblasts (HPO) through a simplified model for primary teeth. The model employed pipette tips filled with 0.037 g of paste, exposed to 185 µL of culture medium for 24 h (n = 6). Release of components was analysed by Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR). HPO were exposed to conditioned media for 24 h. Cell viability was assessed by cell density and metabolic activity, and release of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF) by immunological assay. Physicochemical properties and antimicrobial efficacy were also evaluated. 1H-NMR spectra analysis showed similarity between ZOE, Endoflas®, Calcicur®, and Vitapex® compared to Calen®/ZO and positive control, which showed distinct released components. Calen®/ZO and Calcicur® exhibited high alkaline pH in all periods and showed similar solubility. Calen®/ZO, ZOE, and Vitapex® showed similar flow rate. Calen®/ZO, Calcicur®, and Vitapex® did not exhibit antimicrobial efficacy. Calen®/ZO presented cytotoxicity (p < 0.05). Pastes did not increase IL-6 release compared to control. Apart from Vitapex®, all pastes significantly induced VEGF/bFGF release. Interactive effects among released products may affect biological response to filling pastes. Calcicur®, ZOE, Endoflas® and Calen®/ZO presented good to moderate cytocompatibility, with low impact on pro-inflammatory cytokine release and induction of growth factors of interest to tissue repair. This simplified model, specific for the evaluation of the cytocompatibility of RCPs on primary teeth, suggests how these pastes might contribute to bone repair in clinical situations of apical periodontitis in children.

Saliva NMR-Based Metabolomics in the War Against COVID-19.

COVID-19 is an emergent, worldwide public health concern. Joint efforts have been made by scientific communities of various fields to better understand the mechanisms of action of SARS-CoV-2. The need to understand the pathophysiological fingerprint and pathways of this disease make metabolomics-related approaches an indispensable tool for properly answering concerns relating to disease course. Determination of the metabolomic profile may help to explain the heterogeneous spectra of COVID-19 clinical phenotypes and be useful in monitoring disease progression as well as therapeutic treatments. In this sense, saliva has proven to be a strategic biofluid, owing not only to its appeal as a noninvasive sampling method but also due to the capacity of the virus to invade epithelial cells of the oral mucosa and salivary gland ducts via ACE2 receptors. Accordingly, important changes in metabolism have been described relating to COVID-19, indicating that metabolomics may open new avenues for understanding the pathophysiology of this disease, especially via longitudinal study designs. Thus, we discuss the importance of comprehending the SARS-CoV-2 salivary metabolomic fingerprint and also highlight the situation of Brazil on the frontlines of the war against COVID-19.

Evaluation of polymethyl methacrylate containing chlorhexidine: A randomized, controlled, split-mouth in situ study.

PURPOSE: To evaluate the antimicrobial action and elemental composition of chlorhexidine (CHX) diacetate in acrylic resins based on PMMA (polymethyl methacrylate) in situ. In addition, ex vivo evaluation of the CHX release mechanism was performed over a 14-day period. METHODS: Three discs of PMMA incorporating CHX and three control discs were mounted on individual oral splints and exposed to the oral cavity of 32 participants for 24 hours. The antimicrobial activity was evaluated by the plate count method. In the second test, elemental analysis of the specimens (n = 10) was performed by X-ray fluorescence before and after use of the device. Chlorhexidine release over a 14-day period was evaluated ex vivo in saliva samples collected from five individuals through proton nuclear magnetic resonance spectroscopy ( ¹H NMR) (500 MHz). RESULTS: Bacterial adhesion, evaluated by the plate count method, did not differ between the experimental material and control. (P> 0.05) The presence of the CHX molecule was detected by X-ray fluorescence before and after insertion of discs containing CHX into the oral cavity of participants. With regard to release, CHX was detected in saliva samples for 14 days and highest during the first 24 hours. When partial least squares discriminant analysis (PLS-DA) was applied in ¹H NMR, we observed a greater difference between the test and control groups. CLINICAL SIGNIFICANCE: The sustained release of CHX from PMMA suggests that such materials may be convenient for reducing the development of biofilm on the surface of the material for use in dentures and temporary restorative materials.

Longitudinal Evaluation of Salivary Iga-S in Children with Early Childhood Caries Before and After Restorative Treatment.

Background: Our aim was to compare salivary levels of secretory immunoglobulin A (s-IgA) in children with early childhood caries (ECCG) and those who are caries-free (CFG) and verify these levels in a follow-up period after restorative treatment. Materials and methods: We selected 46 systemically healthy children in the complete primary dentition period, who were allocated into two groups: CFG (n = 23) and ECCG (dmf-s > 0; n = 23). Unstimulated whole saliva was obtained at baseline from both groups and during the follow-up period (7 days, 1, 2 and 3 months) in the ECCG group. The s-IgA was measured using an ELISA assay, and total protein was assessed using the Bradford method. We also evaluated the flow rate (mL/min), Streptococcus mutans and Lactobacillus spp. counting using selective media plaques. The data were submitted to statistical analysis using the software SPSS 20.0 (SPSS Inc, IL, USA) with a confidence interval set at 95%. Results: Salivary s-IgA levels were higher in baseline of ECCG than in CFG (p<0.05). No statistically significant differences were observed between s-IgA salivary levels at baseline and the evaluations after dental treatment in ECCG (p>0.05). However, we observed two different changes in s-IgA levels among participants: one group presented s-IgA reduction, and the other group demonstrated its maintenance. It was shown that patients from the ECCG group who presented a reduction in s-IgA levels during follow-up also showed a decrease in Streptococcus mutans and Lactobacillus spp. count (p<0.05), in contrast to patients who did not present this reduction. The flow rate and total protein were similar between groups (p>0.05). Conclusions: The present data support the idea that children with early childhood caries present higher levels of s-IgA in saliva than caries-free children. The restorative dental treatment does not have a significant influence on salivary levels of this immunoglobulin during the follow-up period.

Conformational Dynamics of a Cysteine-Stabilized Plant Defensin Reveals an Evolutionary Mechanism to Expose Hydrophobic Residues.

Sugar cane defensin 5 (Sd5) is a small antifungal protein, whose structure is held together by four conserved disulfide bridges. Sd5 and other proteins sharing a cysteine-stabilized α-ß (CSαß) fold lack a regular hydrophobic core. Instead, they are stabilized by tertiary contacts formed by surface-exposed hydrophilic and hydrophobic residues. Despite excessive cross-links, Sd5 exhibits complex millisecond conformational dynamics involving all secondary structure elements. We used Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion (RD) measurements performed at different temperatures and denaturant concentrations to probe brief excursions of Sd5 to a sparsely populated "excited" state. Temperature-dependent CPMG RD experiments reveal that the excited state is enthalpically unfavorable, suggesting a rearrangement of stabilizing contacts formed by surface-exposed side chains and/or secondary structure, while the experiments performed at different denaturant concentrations suggest a decrease in accessible surface area of Sd5 in the excited state. The measured backbone 15N chemical shift changes point to a global conformational rearrangement such as a potential α- to ß-transition of the Sd5 α-helix or other major secondary structure reorganization and concomitant conformational changes in other parts of the protein. Overall, the emerging picture of Sd5 dynamics suggests this protein can populate two alternative well-ordered conformational states, with the excited conformer being more compact than the native state and having a distinct secondary structure and side-chain arrangements. The observation of an energetically unfavorable yet more compact excited state reveals a remarkable evolution of the CSαß fold to expose and reorganize hydrophobic residues, which enables the creation of versatile binding sites.

A Dynamic Overview of Antimicrobial Peptides and Their Complexes.

In this narrative review, we comprehensively review the available information about the recognition, structure, and dynamics of antimicrobial peptides (AMPs). Their complex behaviors occur across a wide range of time scales and have been challenging to portray. Recent advances in nuclear magnetic resonance and molecular dynamics simulations have revealed the importance of the molecular plasticity of AMPs and their abilities to recognize targets. We also highlight experimental data obtained using nuclear magnetic resonance methodologies, showing that conformational selection is a major mechanism of target interaction in AMP families.

Structural and Dynamic Insights of the Interaction between Tritrpticin and Micelles: An NMR Study.

A large number of antimicrobial peptides (AMPs) acts with high selectivity and specificity through interactions with membrane lipid components. These peptides undergo complex conformational changes in solution; upon binding to an interface, one major conformation is stabilized. Here we describe a study of the interaction between tritrpticin (TRP3), a cathelicidin AMP, and micelles of different chemical composition. The peptide's structure and dynamics were examined using one-dimensional and two-dimensional NMR. Our data showed that the interaction occurred by conformational selection and the peptide acquired similar structures in all systems studied, despite differences in detergent headgroup charge or dipole orientation. Fluorescence and paramagnetic relaxation enhancement experiments showed that the peptide is located in the interface region and is slightly more deeply inserted in 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-1'-rac-glycerol (LMPG, anionic) than in 1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine (LLPC, zwitterionic) micelles. Moreover, the tilt angle of an assumed helical portion of the peptide is similar in both systems. In previous work we proposed that TRP3 acts by a toroidal pore mechanism. In view of the high hydrophobic core exposure, hydration, and curvature presented by micelles, the conformation of TRP3 in these systems could be related to the peptide's conformation in the toroidal pore.

Salivary Metabolite Fingerprint of Type 1 Diabetes in Young Children.

Metabolomics is an important tool for the evaluation of the human condition, in both health or disease. This study analyzed the salivary components of type I diabetic children (DM1) under six years of age, to assess oral health related to diabetes control, as well as metabolite profiling using NMR. Partial least squared discriminant analysis (PLS-DA) was used to compare healthy (HG) and uncontrolled DM1 subjects that demonstrated a separation between the groups with classificatory performance of ACC = 0.80, R(2) = 0.92, Q(2) = 0.02 and for DM1 children with glycemia >200 mg/dL of ACC = 0.74, R(2) = 0.91, Q(2) = 0.06. The metabolites that mostly contributed to the distinction between the groups in the loading factor were acetate, n-acetyl-sugar, lactate, and sugar. The univariate analysis showed a decreased salivary concentration of succinic acid and increased levels of lactate, acetate, and sucrose in uncontrolled and DM1 children with glycemia >200 mg/dL. The present study demonstrates that the salivary profile of DM1 differs from that of HG children. It appears that diabetes status control has an important effect on the salivary composition.

Dissection of the water cavity of yeast thioredoxin 1: the effect of a hydrophobic residue in the cavity.

The water cavity of yeast thioredoxin 1 (yTrx1) is an ancestral, conserved structural element that is poorly understood. We recently demonstrated that the water cavity is involved in the complex protein dynamics that are responsible for the catalytically relevant event of coupling hydration, proton exchange, and motion at the interacting loops. Its main feature is the presence of the conserved polar residue, Asp24, which is buried in a hydrophobic cavity. Here, we evaluated the role of the solvation of Asp24 as the main element that is responsible for the formation of the water cavity in thioredoxins. We showed that the substitution of Asp24 with a hydrophobic residue (D24A) was not sufficient to completely close the cavity. The dynamics of the D24A mutant of yTrx1 at multiple time scales revealed that the D24A mutant presents motions at different time scales near the active site, interaction loops, and water cavity, revealing the existence of a smaller dissected cavity. Molecular dynamics simulation, along with experimental molecular dynamics, allowed a detailed description of the water cavity in wild-type yTrx1 and D24A. The cavity connects the interacting loops, the central ß-sheet, and α-helices 2 and 4. It is formed by three contiguous lobes, which we call lobes A-C. Lobe A is hydrophilic and the most superficial. It is formed primarily by the conserved Lys54. Lobe B is the central lobe formed by the catalytically important residues Cys33 and Asp24, which are strategically positioned. Lobe C is the most hydrophobic and is formed by the conserved cis-Pro73. The central lobe B is closed upon introduction of the D24A mutation, revealing that independent forces other than solvation of Asp24 maintain lobes A and C in the open configuration. These data allow us to better understand the properties of this enzyme.

Unique properties of human ß-defensin 6 (hBD6) and glycosaminoglycan complex: sandwich-like dimerization and competition with the chemokine receptor 2 (CCR2) binding site.

Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human ß-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the ß2 and ß3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and ß2-ß3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a "sandwich-like" model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding.

Hydration and conformational equilibrium in yeast thioredoxin 1: implication for H(+) exchange.

One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H(+) exchange.

Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire µs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the µs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1.

Beta-parvalbumins from different fish species have been identified as the main elicitors of IgE-mediated reactions in fish-allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross-reactivity among fish parvalbumins. In addition, the high-pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca(2+) -free forms for potential use in immunotherapy.