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Objectives This article evaluated the effect of substrates rigidities on the post-fatigue fracture resistance of adhesively cemented simplified restorations in lithium disilicate glass ceramic. Methods Precrystalized computer-aided design/computer-aided manufacturing ceramic blocks were processed into disc-shaped specimens (n = 10, Ø = 10 mm), mimicking a simplified restoration at two thicknesses (0.5 and 1.0 mm). Thereafter, the discs were cemented onto different base substrates (dentin analogue [control], dentin analogue with a central core build-up of resin composite [RC], or glass ionomer cement [GIC]). The specimens were subjected to mechanical cycling in a chewing simulator (100 N, 1 × 106 cycles, 4 Hz) and then subjected to thermocycling aging (10,000 cycles, 5/37/55°C, 30 seconds). After the fatigue protocol, the specimens were loaded until failure (N) in a universal testing machine. Finite element analysis calculated the first principal stress at the center of the adhesive interface. Results The results showed that “restoration thickness,” “type of substrate,” and their interaction were statistically significant (one-way analysis of variance; p Conclusion More flexible base material reduces the fracture load and increases the stress magnitude of adhesively cemented lithium disilicate restorations regardless the ceramic thickness. Therefore, more rigid substrates are suggested to be used to prevent restoration mechanical failures.

Zika virus (ZIKV) emerged as a global public health concern due to its relationship with severe neurological disorders. Non-structural (NS) proteins of ZIKV are essential for viral replication, regulatory function, and subversion of host responses. NS2B is a membrane protein responsible for the regulation of viral protease activity. This protein has transmembrane domains critical for the localization of viral protease to the endoplasmic reticulum membrane and a hydrophilic domain essential for folding, recruitment, and protease activity. Therefore, NS2B is considered a cofactor of viral protease which processes viral polyprotein and is essential for virus replication, making it an attractive antiviral drug target. Here, we report the backbone 1H, 15N, 13C resonance assignments of the full-length NS2B by high-resolution NMR. The backbone assignment will be necessary for determining the three-dimensional structure and backbone dynamics of NS2B, interaction mapping and screening potential of antiviral drugs against ZIKV and related pathogenic flaviviruses.

To study the influence of peritoneal dialysis (PD) on the salivary metabolite profile of children and adolescents with renal failure. 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, 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. Children and adolescents subjected to PD have a different salivary metabolic profile from that of their healthy subjects. 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.

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

The present study aims to identify the salivary metabolic profile of healthy infants and young children, and to correlate this with age, salivary gland maturation, and dentition. Forty-eight children were selected after clinical evaluation in which all intraoral structures were examined. Total unstimulated saliva was collected, and salivary metabolites were analyzed by 1H Nuclear Magnetic Resonance (NMR) at 25 °C. Partial least squares discriminant analysis (PLS-DA), orthogonal PLS-DA (O-PLS-DA), and univariate analysis were used, adopting a 95% confidence interval. The study showed a distinct salivary metabolomic profile related to age and developmental phase. The saliva of children in the pre-eruption teeth period showed a different metabolite profile than that of children after the eruption. However, more evident changes were observed in the saliva profile of children older than 30 months. Alanine, choline, ethanol, lactate, and sugar region were found in higher levels in the saliva of patients before 30 months old. Acetate, N-acetyl sugar, butyrate, caproate, creatinine, leucine, phenylalanine, propionate, valine, succinate, and valerate were found to be more abundant in the saliva of children after 30 months old. The saliva profile is a result of changes in age and dental eruption, and these findings can be useful for monitoring the physiological changes that occur in infancy.

The purpose of this critical review is to assess if children and adolescents with hyposalivation are more affected by dental caries than those with normal flow rate.A literature search was performed using keywords and MeSH terms related to hyposalivation and dental caries in the Medline/PubMed, Web of Science, Cochrane Library, Scopus, LILACS/BBO databases and in gray literature without language or date restrictions until March 2022. Observational studies that accessed the presence of dental caries in patients up to 18 years-old with hyposalivation and compared with a control group (normal salivation rate) were considered eligible. The results from search were imported to EndNote Web, where duplicates were removed followed by title/abstract and full text analysis.A total of 12,236 non-duplicated studies were found and 14 fulfilled the criteria and were included in the present review, 9 cross-sectional and 5 cohorts. Stimulated salivary flow rates were assessed in 3644 participants, aged 3-17 years. Three cohort and three cross-sectional studies observed association between low salivary flow rates and the presence of dental caries, while the other 9 included articles did not verify this association. However, the absence of a standard criteria for the hyposalivation classification in young patients was observed and brough light to this important limitation among the studies.The salivary flow rate estimation for caries risk assessment must be the target of further studies to make possible and reliable, homogeneous, and unbiasedly assessment of the association between hyposalivation and dental caries in young patients.

Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77Se–1H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1Hβ1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se-Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1Hβ1 and 1Hβ2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1Hβ1 prótons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se – S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77Se. Furthermore the large spectral window of the 77Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.

The aim of this study was to characterize the salivary metabolomic profile in adolescents with juvenile systemic lupus erythematosus (jSLE). A total of 24 adolescents with jSLE (15.92 ± 2.06 years) and 12 systemically healthy controls (15.25 ± 2.7 years) were included in the study. Participants underwent rheumatologic testing and periodontal examination, with the recording of plaque index (PI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing index (BPI). Unstimulated whole saliva was collected from both groups and stored at -80 ºC. The salivary proton nuclear magnetic resonance (1H-NMR) spectra were acquired in a spectrometer operating at 500 MHz. Partial least squared discriminant analysis (PLS-DA) and orthogonal PLS-DA (O-PLS-DA) were used for statistical analysis. Mean CAL and PI were significantly increased in the group with jSLE (p < 0.01). Patients with jSLE presented a significantly different salivary metabolic profile (accuracy = 0.54; R2 = 0.86; Q2 = -0.293), significantly higher salivary levels of N-acetyl sugars, and significantly reduced levels of phenylalanine, glycine, taurine, hydroxybutyrate, and valerate compared with healthy controls (p < 0.05). It is suggested that the salivary metabolomic profile analyzed by 1H NMR in patients with jSLE presents a different fingerprint that the systemically healthy subjects. Integrating the variation of metabolites with the identification of the metabolic pathways involved seems to provide a better understanding of the influence of systemic disease on salivary metabolites.

Zika virus (ZIKV) emerged as a global public health concern due to its relationship with severe neurological disorders. Non-structural (NS) proteins of ZIKV are essential for viral replication, regulatory function, and subversion of host responses. NS2B is a membrane protein responsible for the regulation of viral protease activity. This protein has transmembrane domains critical for the localization of viral protease to the endoplasmic reticulum membrane and a hydrophilic domain essential for folding, recruitment, and protease activity. Therefore, NS2B is considered a cofactor of viral protease which processes viral polyprotein and is essential for virus replication, making it an attractive antiviral drug target. Here, we report the backbone

The aim of the present study was to verify if a protocol for cleaning the oral cavity of infants in the pre-dental period can reduce extrinsic salivary metabolites observed through Nuclear Magnetic Resonance (NMR). A cross-sectional clinical study with a convenience sample was conducted, and infants were recruited at the UFRJ Pediatric Dentistry Clinic. Participants who had used antibiotics and/or antifungals up to 3 months before and whose legal guardians did not consent or sign the Informed Consent Form were excluded. An anamnesis was performed with the guardians and the participants' intraoral clinical examination. Initial collection of unstimulated total saliva was performed using an automatic pipette with sterile plastic tips in the buccal floor region, at least 1 h after the last feeding. Subsequently, the infants' oral mucosa was cleaned with gauze moistened with filtered water, and after 5 min, a new collection was performed, using the same methodology. The obtained samples were immediately transferred on ice to the laboratory, centrifuged (10,000 g), and stored at −80°C. The NMR analyses were performed using a 500-MHz spectrometer Bruker, Germany); evaluations were done via the 1H and 1H-1H TOCSY spectra for metabolite signaling. Eleven pre-dental infants were evaluated, with a mean age of 3.8 months, including six girls (55%). Of these, nine participants (82%) were exclusively breastfed. The higher presence of components such as lactose, glucose, sugars, acetate, alanine, and lactate were observed in the samples before oral mucosa cleaning. Regarding the type of diet, more lactose was observed in the saliva of patients who were exclusively breastfed than those that received mixed feeding. We conclude that the oral mucosa cleaning of infants in the pre-dental period tends to reduce the concentration of extrinsic components from the diet, such as lactose, in the salivary metabolomic profile analyzed by NMR.

Aim: This in vivo experimental study investigated bacterial microbiome and metabolome longitudinal changes associated with enamel caries lesion progression and arrest. Methods: We induced natural caries activity in three caries-free volunteers prior to four premolar extractions for orthodontic reasons. The experimental model included placement of a modified orthodontic band on smooth surfaces and a mesh on occlusal surfaces. We applied the caries-inducing protocol for 4- and 6-weeks, and subsequently promoted caries lesion arrest via a 2-week toothbrushing period. Lesions were verified clinically and quantitated via micro-CT enamel density measurements. The biofilm microbial composition was determined via 16S rRNA gene Illumina sequencing and NMR spectrometry was used for metabolomics. Results: Biofilm maturation and caries lesion progression were characterized by an increase in Gram-negative anaerobes, including Veillonella and Prevotella. Streptococcus was associated caries lesion progression, while a more equal distribution of Streptococcus, Bifidobacterium, Atopobium, Prevotella, Veillonella, and Saccharibacteria (TM7) characterized arrest. Lactate, acetate, pyruvate, alanine, valine, and sugars were more abundant in mature biofilms compared to newly formed biofilms. Conclusions: These longitudinal bacterial microbiome and metabolome results provide novel mechanistic insights into the role of the biofilm in caries progression and arrest and offer promising candidate biomarkers for validation in future studies.

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-β (CSαβ) 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

Background The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function. Methods TcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15N relaxation experiments, and microsecond molecular dynamics (MD) simulations. Results TcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the μs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations. Conclusion Based on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation. General significance Understanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications.

Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D 1H and 2D 15N-1H NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.

Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents sequence homology to TFPI (tissue factor pathway inhibitor). It binds to the coagulation enzyme factor Xa (FXa) or to its zymogen form, FX, and further inhibits tissue factor/FVIIa complex (extrinsic Xnase compex). Differently from TFPI, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. The Ixolaris-FXa/FX complex formation has been characterized by using a combination of biophysical and biochemical technics although no structural data is currently available. In this study, we reported the NMR chemical shift assignment of Ixolaris, as a first step to further establishing the structure, dynamics and function relationship for this protein.

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 (p0.05). 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 (p0.05). 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.

Introduction: Puerperal woman and new-born children are vulnerable and frequently have neglected health conditions. Aim: The objective is to describe the oral health and saliva profiles of women and their babies during the breastfeeding period, including breast milk. Materials and Methods: Forty-seven mothers were interviewed and demographic data were recorded. The mother-baby pairs underwent intraoral examination. The mothers were submitted to examination of oral mucosa, oral hygiene status by O’Leary, periodontal condition and caries (DMF-T: decayed, missing and filled teeth) in order to establish buccal conditions. The babies had their oral mucosa and teeth examined. Salivary samples of babies and mothers as well the breastmilk of mothers were collected and analysed by 1 H-NMR through a 600 MHz spectrometer. The data were analysed in a statistical program SPSS.21 (IBM Statistics). Results: The mothers’ mean age was 27-years-old and 53.9% of mothers were overweight. The oral condition revealed poor oral health: DMF-T=8.20, 72.4% had gingivitis and 62% had dental plaque. The babies presented 4.18% cases of oral candidiasis and 2.08% cases of Bohn nodules, with no caries. The salivary 1 H-NMR spectra from babies with more than six months of age showed increased levels of lactate, ethanol, acetate, propionate, N-butyrate and N-acetyl sugars and reduced levels of other sugars. The 1 H-NMR analysis of salivary samples from the mothers showed metabolites such as propionate, ethanol, lactate, acetate, butyrate and N-acetyl and sugar region. The 1 H-NMR breast milk demonstrated high quantity of lactose in a region of spectra characteristic from sugars. It was concluded that the mothers had low levels caries activity; however, though they had past dental caries history. This may have an impact on the oral health of their children. Conclusion: Our study focused on the oral health and saliva profiles of women and their babies during breastfeeding period. This data could design a preventive programme that would improve the oral health and quality of life.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior FINEP - Financiadora de Estudos e Projetos, Financiadora de Estudos e Projetos FAPERJ - Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro In Brazil and in other tropical areas Zika virus infection was directly associated with clinical complications as microcephaly in newborn children whose mothers were infected during pregnancy and the Guillain-Barré syndrome in adults. Recently, research has been focused on developing new vaccines and drug candidates against Zika virus infection since none of those are available. In order to contribute to vaccine and drug development efforts, it becomes important the understanding of the molecular basis of the Zika virus recognition, infection and blockade. To this purpose, it is essential the structural determination of the Zika virus proteins. The genome sequencing of the Zika virus identified ten proteins, being three structural (protein E, protein C and protein prM) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Together, these proteins are the main targets for drugs and antibody recognition. Here we examine new discoveries on high-resolution structural biology of Zika virus, observing the interactions and functions of its proteins identified via state-of-art structural methodologies as X-ray crystallography, nuclear magnetic resonance spectroscopy and cryogenic electronic microscopy. The aim of the present study is to contribute to the understanding of the structural basis of Zika virus infection at an atomic level and to point out similarities and differences to others flaviviruses.

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.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven β-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

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 β-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.

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X-ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. Copyright © 2015 John Wiley & Sons, Ltd.

We report the preparation, characterization, and drug release kinetics of a pH-responsive hydrogel film from a dendrimer megamer. The megamer (GP32) is a three-dimensional reticulated structure with a mean diameter of 71.16 nm (PDI 0.150) and was prepared by the reaction between Poly(amidoamine) generation4 (PAMAM G4) dendrimer and glutaraldehyde (G:P molar ratio 32). The crosslinking units in the megamer are provided mainly by the bicyclic dimer 2-hydroxy-3,4,4a,7,8,8a–hexahydro-2H-chromene-6-carbaldehyde as determined by high-resolution (800 MHz) 1H NMR and FTIR. The hydrogel film (F[GP32]) is formed upon evaporation of a methanolic solution of the megamer and has a high degree of organization and homogeneity. Further crosslinking with glutaraldehyde (CLF[GP32]) enhanced the mechanical properties of the hydrogel film. The chemical constitution and unique megamer architecture enable the hydrogel film to carry both lipophilic and hydrophilic substances. The film did not cause any dermal irritation or clinical signs of toxicity in tests on rabbits, allowed for a sustained release of ketoprofen and played an important role in the process of drug delivery into the receptor medium. This performance taken together with the absence of toxicity makes this hydrogel film a good choice for dermal sustained drug release.

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer–dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218–31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218–31 complexes. NMR relaxation measurements indicated that residues around the CCR218–31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/C...

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 Ca2+-free forms for potential use in immunotherapy. Proteins 2014; 82:3032–3042. © 2014 Wiley Periodicals, Inc.

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 β-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 β2 and β3 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 β2-β3 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.

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.

Background:The purpose of this study was to evaluate the effect of different bonding agents on bond-strength to demineralized enamel after remineralizing treatments and resin infiltration.Methods:Buccal enamel of 120 bovine incisors was polished and then were divided into five experimental groups: SE (sound enamel); DE (demineralized enamel); AS (demineralized enamel immersed in artificial saliva for eight weeks); NaF (demineralized enamel treated with 0.05% sodium fluoride solution (one minute) for eight weeks); Ic (demineralized enamel infiltrated with a low-viscosity resin (Icon-DGM). These groups were subdivided according to adhesive system used: self-etching adhesive Adper Easy One (3M/ESPE) and etch-and-rinse adhesive Single Bond (3M/ESPE). The composite resin blocks were fabricated using a Teflon matrix. A thermomechanical cycling machine was used to carry out the artificial aging of the specimens and thus were sectioned into sticks. The microtensile tests were performed using a universal testing machine at a cross-head speed of 1 mm/min. Data (in MPa) were subjected to two-way ANOVA and Tukey’s tests (5%).Results: Significant differences were found for both factors tested and interactions (pConclusions:Resin infiltration did not affect the bond strength of demineralized enamel for both adhesive systems tested. For etch-and-rinse adhesive, no differences were observed for the tested groups. For self-etching adhesive, only the resin-infiltrated group showed similar bond strength to sound enamel. Both etch-and-rinse and self-etching adhesive systems can be used in resin-infiltrated enamel, if a composite restoration needs to be further performed. In enamel that has undergone the de/remineralization process, the use of a total-etch adhesive might be preferable for the restorative procedure.

Antimicrobial peptides are highly dynamic entities that acquire structure upon binding to a membrane interface. To better understand the structure and the mechanism for the molecular recognition of dodecylphosphocholine (DPC) micelles by the anticoccidial peptide PW2, we performed molecular dynamics (MD) simulations guided by NMR experimental data, focusing on strategies to explore the transient nature of micelles, which rearrange on a millisecond to second timescale. We simulated the association of PW2 with a pre-built DPC micelle and with free-DPC molecules that spontaneously forms micelles in the presence of the peptide along the simulation. The simulation with spontaneous micelle formation provided the adequate environment which replicated the experimental data. The unrestrained MD simulations reproduced the NMR structure for the entire 100 ns MD simulation time. Hidden discrete conformational states could be described. Coulomb interactions are important for initial approximation and hydrogen bonds for anchoring the aromatic region at the interface, being essential for the stabilization of the interaction. Arg9 is strongly attached with phosphate. We observed a helix elongation process stabilized by the intermolecular peptide-micelle association. Full association that mimics the experimental data only happens after complete micelle re-association. Fast micelle dynamics without dissociation of surfactants leads to only superficial binding.

We demonstrate for the first time a complete small protein characterization with the projection-decomposition approach, including full assignments as well as determination of the 3D fold. In TOCSY- and NOESY-type 4D experiments, pairing of signals from hydrogens and from their respective heavy atoms in decompositions represents a new problem. An approach, referred to as "DIADECOMP" (diagonal decomposition), is introduced to solve this problem; it consists of two separate decompositions of the input projections, differing in a 45° rotation of the spectral axes. While DIADECOMP requires a somewhat complex formulation, in practice it results in observing signals in the rotated decompositions that correspond to sums or differences of frequencies. When applied to a small protein, human defensin β6, the analysis of a HCC(CO)NH-TOCSY with DIADECOMP results in largely unambiguous assignments of the aliphatic side chain groups. Furthermore, DIADECOMP applied to a 15N-HSQC-NOESY-15N-HSQC provides all expected short distances between amide groups (defined as all HN-HN distances

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.

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.

Gad m 1 is the major allergen from Atlantic cod. It belongs to β-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. β-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of β-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a β-parvalbumin: Gad m 1. This report includes: 1H, 13C and 15N resonance assignments of Gad m 1 as well as the second structure information based on the 13C chemical shifts.

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.

A polysaccharide fraction containing acidic galactan (AG) was obtained from eggs of the mollusc Pomacea lineata by precipitation with acetone. Its structure was investigated using a combination of chemical analysis, intrinsic viscosity and NMR spectroscopy methods, including 1D and 2D, COSY, TOCSY and HSQC experiments. The chemistry analysis showed that the acidic galactan did not possess neither sulfate nor uronic acid. The intrinsic and relative viscosities were 0.44 ± 0.05 and 1.744 ± 0.07 dL.g −1 , respectively. The NMR confirmed that the acidic galactan had a backbone containing β- d -Gal is a predominant compound and presence of the β- d -GlcNAc in less proportion. In addition, were found pyruvate groups forming six-membered cyclic ketals as 4,6- O -(1′carboxy)-ethylidene-β- d -galactose residues with configuration R in some β- d -Gal p .

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. 1H-15N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

Phylloseptins are antimicrobial peptides of 19–20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2 ≥ PS-1 > PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions.

Keywords NMR Saccharomyces cerevisiae Structure Thioredoxin 2 YeastAbbreviationsTrx thioredoxinBiological contextThioredoxins (Trxs) are ubiquitous heat-stable pro-teins of molecular mass approximately 12,000 Da thatfunction as dithiol oxidoreductases, and are impor-tant for cellular defense against oxidative stress(Monje-Casas et al. 2004). Trxs act as hydrogen do-nors for reductive enzymes such as ribonucleotidereductases (Laurent et al. 1964) and thioredoxinperoxidases (Kang et al. 1998), as a general reductantfor disulfides in proteins (Holmgren 1985), and asregulatory factors for enzymes or receptors inphotosynthetic systems (Buchanan 1991). They alsopromote DNA binding of transcription factors, suchas NF-jB and YAP-1 (Schenk et al. 1994), amongothers.In yeast, there are two cytoplasmic (Trx1 and Trx2)and one mitochondrial (Trx3) isoforms. Some authorsconsider that the preservation of the two cytoplasmicisoforms during evolution is not related to function,meaning that they are redundant. Recently, Monje-Casas et al. (2004) described some differences inexpression levels under normal and oxidative stressconditions. The basal level of both thioredoxins is thesame, but becomes different after treatment withhydrogen peroxide. Trx2 shows an increase of 14 foldin its expression, whereas Trx1 remains unaffected.Gene duplication is widespread, but it is not alwayspreserved in evolution. In Saccharomyces cerevisiaethere might be an explanation for the preservation ofboth Trx1 and Trx2. The parallel evolution of yeastthioredoxins occurred possibly because they exerteddifferent regulatory function. Since regulation occursthrough binding to different cellular targets, Trx1 andTrx2 might be specialized to bind to different targets.Three-dimensional structures of several thioredoxinshave been determined. We have determined the NMRstructure of the two cytoplasmic isoforms. In this note,we report the structure of thioredoxin 2 (PDB ID:2HSY) and compare it to other thioredoxins.Methods and resultsExpression and purificationIndividual colonies of the transformants were grown inLB medium containing 100 lg/ml ampicillin at 37 Cfor

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the (1)H, (15)N and (13)C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.

A new steroidal saponin was isolated from the leaves of Agave shrevei. Its structure was established as 3-[O-beta-D-glucopyranosyl-(1--2)-O-[O-beta-D-glucopyranosyl-(1--4)-O-[O-beta-D-glucopyranosyl-(1--6)]-O-beta-D-glucopyranosyl-(1--4)-beta-D-galactopyranosyl)-oxy]-(3beta,5alpha,25R)-spirostane. The structural identification was performed using detailed analyses of 1H- and 13C-NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, HMBC, and HMQC) and chemical conversions. The haemolytic activity of the steroidal saponin was evaluated using an in vitro assay.

Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis.

With the advent of structural genomics, the need for fast structural information about unknown proteins has increased. We describe a new methodology, based on 13C, 15N and ¹H chemical shift dispersion to predict the amount of secondary structure of unassigned proteins from their 15N- and/or 13C-edited heteronuclear single quantum coherence (HSQC) spectra. This methodology has been coded into a software called PASSNMR (Prediction of the Amount of Secondary Structure by Nuclear Magnetic Resonance), which can be accessed directly from the Internet. PASSNMR program is a powerful tool for screening proteins for proteomic or structural genomic investigations when used with recent methodologies that take advantage of the use of the antibiotic rifampicin to selectively label the heterologous proteins expressed in E. coli. PASSNMR analysis can be useful as a first approach to predict the amount of secondary structure in proteins to structural genomics. Information about the secondary structure of proteins can be obtained even before protein purification, with small quantities of protein, just by performing two simple nuclear magnetic resonance (NMR) experiments and using PASSNMR program.

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of β-amyloid peptide (Aβ). Here we describe the stabilization of small oligomers of Aβ by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate). The experiments were performed with either Aβ-1-42 or with Aβ-13-23, a shorter version of Aβ that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Aβ aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Aβ forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Aβ-13-23 in SDS micelles and found features of an α-helix from Lys16 to Phe20. 1H TOCSY spectra of Aβ-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.

A linear sulfated fucan with a regular repeating sequence of [3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2,4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]][subscript n] is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by heparin cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with heparin cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by heparin cofactor II. Sulfated fucan with [approximately] 45 tetrasaccharide repeating units binds to heparin cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with [approximately] 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of heparin cofactor II.

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.

We applied a high-throughput strategy for the screening of targets for structural proteomics of Xanthomonas axonopodis pv citri. This strategy is based on the rapid 1H–15N HSQC NMR analysis of bacterial lysates containing selectively 15N-labelled heterologous proteins. Our analysis permitted us to classify the 19 soluble candidates in terms of ‘foldedness’, that is, the extent to which they present a well-folded solution structure, as reflected by the quality of their NMR spectra. This classification allowed us to define a priority list to be used as a guide to select protein candidates for further structural studies.