RESUMO
Bacterial cell division proteins, especially the tubulin homologue FtsZ, have emerged as strong targets for developing new antibiotics. Here, we have utilized the fission yeast heterologous expression system to develop a cell-based assay to screen for small molecules that directly and specifically target the bacterial cell division protein FtsZ. The strategy also allows for simultaneous assessment of the toxicity of the drugs to eukaryotic yeast cells. As a proof-of-concept of the utility of this assay, we demonstrate the effect of the inhibitors sanguinarine, berberine, and PC190723 on FtsZ. Though sanguinarine and berberine affect FtsZ polymerization, they exert a toxic effect on the cells. Further, using this assay system, we show that PC190723 affects Helicobacter pylori FtsZ function and gain new insights into the molecular determinants of resistance to PC190723. On the basis of sequence and structural analysis and site-specific mutations, we demonstrate that the presence of salt bridge interactions between the central H7 helix and ß-strands S9 and S10 mediates resistance to PC190723 in FtsZ. The single-step in vivo cell-based assay using fission yeast enabled us to dissect the contribution of sequence-specific features of FtsZ and cell permeability effects associated with bacterial cell envelopes. Thus, our assay serves as a potent tool to rapidly identify novel compounds targeting polymeric bacterial cytoskeletal proteins like FtsZ to understand how they alter polymerization dynamics and address resistance determinants in targets.
Assuntos
Berberina , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Antibacterianos/farmacologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.
Assuntos
Actinas , Proteínas de Bactérias , Nucleotídeos , Spiroplasma citri , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Nucleotídeos/metabolismo , Spiroplasma citri/genética , Spiroplasma citri/metabolismoRESUMO
Stable maintenance and partitioning of the 'Fertility' plasmid or the F plasmid in its host Escherichia coli require the function of a ParA superfamily of proteins known as SopA. The mechanism by which SopA mediates plasmid segregation is well studied. SopA is a nucleoid-binding protein and binds DNA in an ATP-dependent but sequence non-specific manner. ATP hydrolysis stimulated by the binding of the SopBC complex mediates the release of SopA from the nucleoid. Cycles of ATP-binding and hydrolysis generate an ATPase gradient that moves the plasmid through a chemophoresis force. Nucleoid binding of SopA thus assumes a central role in its plasmid-partitioning function. However, earlier work also suggests that the F plasmid can be partitioned into anucleate cells, thus implicating nucleoid independent partitioning. Interestingly, SopA is also reported to be associated with the inner membrane of the bacteria. Here, we report the identification of a possible membrane-targeting sequence, a predicted amphipathic helix, at the C-terminus of SopA. Molecular dynamics simulations indicate that the predicted amphipathic helical motif of SopA has weak affinity for membranes. Moreover, we experimentally show that SopA can associate with bacterial membranes, is detectable in the membrane fractions of bacterial lysates, and is sensitive to the membrane potential. Further, unlike the wild-type SopA, a deletion of the C-terminal 29 amino acids results in the loss of F plasmids from bacterial cells.
Assuntos
Proteínas de Escherichia coli , Fator F , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genéticaRESUMO
The data presented in this paper supports the research article "A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment" ( Vutukuru et al., 2016) [1]. We compared a list of sepsis causing pathogens to the ApoH binding data given to us by ApoH technologies. The data highlights the binding of ApoH beads to sepsis causing pathogens.
RESUMO
BACKGROUND: Antigenicity of HIV-1 envelope proteins (Envs) of both lab-adapted and primary isolates expressed on the cell surface rarely match with in vitro neutralization of viruses, pseudo-typed with corresponding Envs. Often, both neutralizing and non-neutralizing antibodies bind to Envs expressed on the cell membrane. This could be due to the lack of efficient cleavage of Env expressed on the cell surface. Naturally occurring, efficiently cleaved Envs with appropriate antigenic properties are relatively rare. Given viral diversity it is essential to increase the pool of candidate Envs suitable for immunogen design. Previously, it has been reported that JRFL Env is the only clade B Env, which is efficiently cleaved on the cell surface and retains desirable antigenic properties. JRCSF is a clade B Env isolated from the same patient as JRFL. JRCSF Env has not been explored aggressively for designing immunogen as the binding characteristics of JRCSF Env to broadly neutralizing antibodies on the cell surface and its cleavage status are unknown. RESULTS: Although JRCSF preferentially binds to most of the other gp120-directed neutralizing antibodies and cleavage dependent antibody, PGT151 efficiently, it binds poorly to CD4-binding-site-directed (CD4-bs-directed) neutralizing antibodies on cell surface. Membrane bound form of modified JRCSF Env containing the N197D mutation binds to CD4-bs-directed neutralizing antibodies better than JRFL, without debilitating its ability to bind quaternary epitope-directed neutralizing antibodies or exposing the CD4i antibody epitopes. In comparison to JRFL (E168K), JRCSF Env binds more efficiently to PG9/PGT145 class of V1/V2-directed conformational antibodies. Biochemical, cell surface staining and gp120 shedding experiments suggest that JRCSF is efficiently cleaved on the cell surface. CONCLUSIONS: Binding of JRCSF Env expressed on cell surface to the various HIV-1 Env-directed antibodies has not been reported earlier. Here, for the first time, we report that compared to JRFL, JRCSF displays epitopes for a larger number of broadly neutralizing antibodies and is also efficiently cleaved when expressed on the cell surface. Thus, considering the diversity of viral Envs and the discovery of conformation dependent glycan-directed antibodies in HIV-1 infected individuals, an innately cleaved JRCSF Env as present on the viral membrane and displaying those distinct epitopes may be an important candidate for immunogen design.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Ligação Proteica , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200µL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
Assuntos
Patógenos Transmitidos pelo Sangue/isolamento & purificação , Sangue/microbiologia , Técnicas Microbiológicas/métodos , Patologia Molecular/métodos , Sepse/diagnóstico , Sepse/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Candida tropicalis/genética , Candida tropicalis/isolamento & purificação , Células Cultivadas , Contagem de Colônia Microbiana/métodos , DNA Bacteriano/sangue , DNA Fúngico/sangue , Enterococcus/genética , Enterococcus/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Fungos/patogenicidade , Genoma Humano , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Sepse/sangue , Fatores de Tempo , beta 2-Glicoproteína I/administração & dosagemRESUMO
Using anti-human CD45 antibody coated beads, we show a 98% reduction of WBCs from spiked blood samples in 1h, thereby enriching it for pathogens. This enrichment allowed the detection of <10CFU of Escherichia coli in 1mL blood using quantitative PCR; something not observed in unenriched samples.
Assuntos
Bactérias/isolamento & purificação , Sangue/microbiologia , Escherichia coli/isolamento & purificação , Separação Imunomagnética/métodos , Leucócitos , Reação em Cadeia da Polimerase/métodos , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/diagnóstico , Humanos , Antígenos Comuns de Leucócito/imunologia , Limite de Detecção , Sensibilidade e EspecificidadeRESUMO
GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca(2+) is a weak agonist of PTH1R, and Ca(2+) in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.
Assuntos
Cálcio/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Nanoestruturas/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Sítios de Ligação , Cálcio/farmacologia , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Receptor Tipo 1 de Hormônio Paratireóideo/agonistas , Receptor Tipo 1 de Hormônio Paratireóideo/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Sialic acid-recognizing Ig-like lectins (Siglecs) are signaling receptors that modulate immune responses, and are targeted for interactions by certain pathogens. We describe two primate Siglecs that were rendered nonfunctional by single genetic events during hominin evolution after our common ancestor with the chimpanzee. SIGLEC13 was deleted by an Alu-mediated recombination event, and a single base pair deletion disrupted the ORF of SIGLEC17. Siglec-13 is expressed on chimpanzee monocytes, innate immune cells that react to bacteria. The human SIGLEC17P pseudogene mRNA is still expressed at high levels in human natural killer cells, which bridge innate and adaptive immune responses. As both resulting pseudogenes are homozygous in all human populations, we resurrected the originally encoded proteins and examined their functions. Chimpanzee Siglec-13 and the resurrected human Siglec-17 recruit a signaling adapter and bind sialic acids. Expression of either Siglec in innate immune cells alters inflammatory cytokine secretion in response to Toll-like receptor-4 stimulation. Both Siglecs can also be engaged by two potentially lethal sialylated bacterial pathogens of newborns and infants, agents with a potential impact on reproductive fitness. Neanderthal and Denisovan genomes show human-like sequences at both loci, corroborating estimates that the initial pseudogenization events occurred in the common ancestral population of these hominins. Both loci also show limited polymorphic diversity, suggesting selection forces predating the origin of modern humans. Taken together, these data suggest that genetic elimination of Siglec-13 and/or Siglec-17 represents signatures of infectious and/or other inflammatory selective processes contributing to population restrictions during hominin origins.
Assuntos
Evolução Molecular , Inativação Gênica , Lectinas/genética , Animais , Deleção de Genes , Humanos , Sistema Imunitário , Primatas , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
We previously reported a human-specific gene conversion of SIGLEC11 by an adjacent paralogous pseudogene (SIGLEC16P), generating a uniquely human form of the Siglec-11 protein, which is expressed in the human brain. Here, we show that Siglec-11 is expressed exclusively in microglia in all human brains studied-a finding of potential relevance to brain evolution, as microglia modulate neuronal survival, and Siglec-11 recruits SHP-1, a tyrosine phosphatase that modulates microglial biology. Following the recent finding of a functional SIGLEC16 allele in human populations, further analysis of the human SIGLEC11 and SIGLEC16/P sequences revealed an unusual series of gene conversion events between two loci. Two tandem and likely simultaneous gene conversions occurred from SIGLEC16P to SIGLEC11 with a potentially deleterious intervening short segment happening to be excluded. One of the conversion events also changed the 5' untranslated sequence, altering predicted transcription factor binding sites. Both of the gene conversions have been dated to ~1-1.2 Ma, after the emergence of the genus Homo, but prior to the emergence of the common ancestor of Denisovans and modern humans about 800,000 years ago, thus suggesting involvement in later stages of hominin brain evolution. In keeping with this, recombinant soluble Siglec-11 binds ligands in the human brain. We also address a second-round more recent gene conversion from SIGLEC11 to SIGLEC16, with the latter showing an allele frequency of ~0.1-0.3 in a worldwide population study. Initial pseudogenization of SIGLEC16 was estimated to occur at least 3 Ma, which thus preceded the gene conversion of SIGLEC11 by SIGLEC16P. As gene conversion usually disrupts the converted gene, the fact that ORFs of hSIGLEC11 and hSIGLEC16 have been maintained after an unusual series of very complex gene conversion events suggests that these events may have been subject to hominin-specific selection forces.
Assuntos
Evolução Molecular , Hominidae/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Regiões 5' não Traduzidas/genética , Adulto , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Conversão Gênica/genética , Frequência do Gene/genética , Loci Gênicos/genética , Genética Populacional , Humanos , Ligantes , Camundongos , Microglia/metabolismo , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Pseudogenes/genética , Alinhamento de Sequência , Fatores de TranscriçãoRESUMO
The primate SIGLEC12 gene encodes one of the CD33-related Siglec family of signaling molecules in immune cells. We had previously reported that this gene harbors a human-specific missense mutation of the codon for an Arg residue required for sialic acid recognition. Here we show that this R122C mutation of the Siglec-XII protein is fixed in the human population, i.e. it occurred prior to the origin of modern humans. Additional mutations have since completely inactivated the SIGLEC12 gene in some but not all humans. The most common inactivating mutation with a global allele frequency of 58% is a single nucleotide frameshift that markedly shortens the open reading frame. Unlike other CD33-related Siglecs that are primarily found on immune cells, we found that Siglec-XII protein is expressed not only on some macrophages but also on various epithelial cell surfaces in humans and chimpanzees. We also found expression on certain human prostate epithelial carcinomas and carcinoma cell lines. This expression correlates with the presence of the nonframeshifted, intact SIGLEC12 allele. Although SIGLEC12 allele status did not predict prostate carcinoma incidence, restoration of expression in a prostate carcinoma cell line homozygous for the frameshift mutation induced altered regulation of several genes associated with carcinoma progression. These stably transfected Siglec-XII-expressing prostate cancer cells also showed enhanced growth in nude mice. Finally, monoclonal antibodies against the protein were internalized by Siglec-XII-expressing prostate carcinoma cells, allowing targeting of a toxin to such cells. Polymorphic expression of Siglec-XII in humans thus has implications for prostate cancer biology and therapeutics.
Assuntos
Regulação Neoplásica da Expressão Gênica , Lectinas/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , Pseudogenes , Alelos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Frequência do Gene , Humanos , Lectinas/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Transplante HeterólogoRESUMO
Optical detection of glucose, high drug loading capacity, and self-regulated drug delivery are simultaneously possible using a multifunctional hybrid nanogel particle under a rational design in a colloid chemistry method. Such hybrid nanogels are made of Ag nanoparticle (NP) cores covered by a copolymer gel shell of poly(4-vinylphenylboronic acid-co-2-(dimethylamino)ethyl acrylate) [p(VPBA-DMAEA)]. The introduction of the glucose sensitive p(VPBA-DMAEA) gel shell onto Ag NPs makes the polymer-bound Ag NPs responsive to glucose. While the small sized Ag cores (10 +/- 3 nm) provide fluorescence as an optical code, the responsive polymer gel shell can adapt to a surrounding medium of different glucose concentrations over a clinically relevant range (0-30 mM), convert the disruptions in homeostasis of glucose level into optical signals, and regulate release of preloaded insulin. This shows a new proof-of-concept for diabetes treatment that exploits the properties from each building block of a multifunctional nano-object. The highly versatile multifunctional hybrid nanogels could potentially be used for simultaneous optical diagnosis, self-regulated therapy, and monitoring of the response to treatment.
Assuntos
Técnicas Biossensoriais/métodos , Glucose/análise , Insulina/metabolismo , Nanotecnologia/métodos , Fenômenos Ópticos , Polietilenoglicóis/química , Polietilenoimina/química , Acrilatos/química , Animais , Ácidos Borônicos/química , Células COS , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Medições Luminescentes , Nanopartículas Metálicas/química , Metilaminas/química , Nanogéis , Tamanho da Partícula , Transição de Fase , Polietilenoglicóis/toxicidade , Polietilenoimina/toxicidade , Prata/química , TemperaturaRESUMO
N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.
Assuntos
Glicoproteínas/fisiologia , Monofenol Mono-Oxigenase/fisiologia , Sequência de Aminoácidos , Animais , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Crystal structures of peanut lectin complexed with Galbeta1-3Gal, methyl-T-antigen, Galbeta1-6GalNAc, Galalpha1-3Gal and Galalpha1-6Glc and that of a crystal grown in the presence of Galalpha1-3Galbeta1-4Gal have been determined using data collected at 100 K. The use of water bridges as a strategy for generating carbohydrate specificity was previously deduced from the complexes of the lectin with lactose (Galbeta1-4Glc) and T-antigen (Galbeta1-3GalNAc). This has been confirmed by the analysis of the complexes with Galbeta1-3Gal and methyl-T-antigen (Galbeta1-3GalNAc-alpha-OMe). A detailed analysis of lectin-sugar interactions in the complexes shows that they are more extensive when the beta-anomer is involved in the linkage. As expected, the second sugar residue is ill-defined when the linkage is 1-->6. There are more than two dozen water molecules which occur in the hydration shells of all structures determined at resolutions better than 2.5 A. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin-sugar interactions are also substantially conserved. The lectin molecule is fairly rigid and does not appear to be affected by changes in temperature.
Assuntos
Dissacarídeos/química , Modelos Moleculares , Aglutinina de Amendoim/química , Dissacarídeos/metabolismo , Aglutinina de Amendoim/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Glycosylation, particularly N-linked glycosylation, profoundly affects protein folding, oligomerization and stability. The increased efficiency of folding of glycosylated proteins could be due to the chaperone-like activity of glycans, which is observed even when the glycan is not attached to the protein. Covalently linked glycans could also facilitate oligomerization by mediating inter-subunit interactions in the protein or stabilizing the oligomer in other ways. Glycosylation also affects the rate of fibril formation in prion proteins: N-glycans reduce the rate of fibril formation, and O-glycans affect the rate either way depending on factors such as position and orientation. It has yet to be determined whether there is any correlation among the sites of glycosylation and the ensuing effect in multiply glycosylated proteins. It is also not apparent whether there is a common pattern in the conservation of glycans in a related family of glycoproteins, but it is evident that glycosylation is a multifaceted post-translational modification. Indeed, glycosylation serves to "outfit" proteins for fold-function balance.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Dobramento de Proteína , Animais , Humanos , Modelos Biológicos , Ligação Proteica , Desnaturação ProteicaRESUMO
These studies attempt to characterize the molten globule-like intermediate in the unfolding pathway of peanut agglutinin (PNA). PNA is the only known example of a homotetrameric protein that lacks the 2,2,2 or the fourfold symmetry. Previous studies have shown that PNA describes a non two-state unfolding process populated with a clearly defined intermediate. The intermediate is monomeric and has lost most of its tertiary structure and has a substantial amount of secondary structure still intact, thus described as a molten-globule (MG)-like intermediate. It was also shown by isothermal titration calorimetry to bind to lactose and some other ligands with an affinity similar to that of the native protein. This paper describes limited protease cleavage experiments on the intermediate using trypsin and protease V8 for its structural characterization. There are two hydrophobic cores in the PNA subunit. These experiments suggest that in the MG-like intermediate, the second hydrophobic core, near the sugar-binding loop of the protein loosens up. This effect is significantly reduced by the presence of 90% saturating lactose, as deduced by a reduction in cleavage propensity. This is also supported by the gain in the tertiary structure as observed by near-UV CD.
Assuntos
Aglutinina de Amendoim/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Desnaturação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Serina Endopeptidases/metabolismo , Espectrofotometria Ultravioleta , Tripsina/metabolismoRESUMO
Synthetic cluster glycosides have often been used to unravel mechanisms of carbohydrate-protein interactions. Although synthetic cluster glycosides are constituted on scaffolds to achieve high avidities in lectin binding, there have been no known attempts to modulate the orientations of the sugar clusters with the aid of a functional scaffold onto which the sugar units are linked. Herein, we describe synthesis, physical, and lectin-binding studies of a series of alpha-D-mannopyranoside and beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside glycoclusters that are attached to a photoswitchable azobenzenoid core. These glycoclusters were synthesized by the amidation of amine-tethered glycopyranosides with azobenzene carbonyl chlorides. From kinetic studies, the cis forms of the azobenzene-glycopyranoside derivative were found to be more stable in aqueous solutions than in organic solvents. Molecular modeling studies were performed to estimate the relative geometries of the photoswitchable glycoclusters in the trans- and cis-isomeric forms. Isothermal titration calorimetry (ITC) was employed to assess the binding of these glycoclusters to lectins peanut agglutinin (PNA) and concanavalin A (Con A). Although binding affinities were enhanced several orders higher as the valency of the sugar was increased, a biphasic-binding profile in ITC plots was observed during few glycoclusters lectin-binding processes. The biphasic-binding profile indicates a "cooperativity" in the binding process. An important outcome of this study is that in addition to inherent clustering of the sugar units as a molecular feature, an induced clustering emanates because of the isomerization of the trans form of the azobenzene scaffold to the cis-isomeric form.
Assuntos
Compostos Azo/química , Carboidratos/química , Concanavalina A/química , Glicosídeos/química , Modelos Moleculares , Aglutinina de Amendoim/química , Compostos Azo/síntese química , Compostos Azo/metabolismo , Configuração de Carboidratos , Metabolismo dos Carboidratos , Concanavalina A/metabolismo , Glicosídeos/síntese química , Glicosídeos/metabolismo , Técnicas de Sonda Molecular , Aglutinina de Amendoim/metabolismo , Fotoquímica , Ligação ProteicaRESUMO
The unfolding pathway of two very similar tetrameric legume lectins soybean agglutinin (SBA) and Concanavalin A (ConA) were determined using GdnCl-induced denaturation. Both proteins displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provided values for conformational stability of the two proteins. It was found that the DeltaG of unfolding of SBA was much higher than ConA at all the temperatures at which the experiments were done. ConA had a T(g) 18 degrees C less than SBA. The higher conformational stability of SBA in comparison to ConA is largely due to substantial differences in their degrees of subunit interactions. Ionic interactions at the interface of the two proteins especially at the noncanonical interface seem to play a significant role in the observed stability differences between these two proteins. Furthermore, SBA is a glycoprotein with a GlcNac2Man9 chain attached to Asn-75 of each subunit. The sugar chain in SBA lies at the noncanonical interface of the protein, and it is found to interact with the amino acid residues in the adjacent noncanonical interface. These interactions further stabilize SBA with respect to ConA, which is not glycosylated.
Assuntos
Aglutininas/química , Canavalia/metabolismo , Concanavalina A/química , Glycine max/metabolismo , Modelos Químicos , Aglutininas/análise , Sítios de Ligação , Simulação por Computador , Concanavalina A/análise , Dimerização , Guanidina/química , Complexos Multiproteicos/análise , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Relação Estrutura-AtividadeRESUMO
It is well known that the sequence of amino acids in proteins code for its tertiary structure. It is also known that there exists a relationship between sequence and the quaternary structure of proteins. The question addressed here is whether the nature of quaternary association can be predicted from the sequence, similar to the three-dimensional structure prediction from the sequence. The class of proteins called legume lectins is an interesting model system to investigate this problem, because they have very high sequence and tertiary structure homology, with diverse forms of quaternary association. Hence, we have used legume lectins as a probe in this paper to (1) gain novel insights about the relationship between sequence and quaternary structure; (2) identify the sequence motifs that are characteristic of a given type of quaternary association; and (3) predict the quaternary association from the sequence motif.
Assuntos
Simulação por Computador , Lectinas/química , Plantas/química , Estrutura Quaternária de Proteína , Software , Homologia Estrutural de Proteína , Algoritmos , Sequência de Aminoácidos , Biologia Computacional , Dados de Sequência Molecular , Proteínas de Plantas/química , Alinhamento de SequênciaRESUMO
Hyaluronan-binding protein 1 (HABP1) is a trimeric protein with high negative charges distributed asymmetrically along the faces of the molecule. Recently, we have reported that HABP1 exhibits a high degree of structural flexibility, which can be perturbed by ions under in vitro conditions near physiological pH (Jha, B. K., Salunke, D. M., and Datta, K. (2003) J. Biol. Chem. 278, 27464-27472). Here, we report the effect of ionic strength and pH on thermodynamic stability of HABP1. Trimeric HABP1 was shown to unfold reversibly upon dissociation ruling out the possibility of existence of folded monomer. An increase in ionic concentration (0.05-1 M) or decrease in pH (pH 8.0-pH 5.0) induced an unusually high thermodynamic stability of HABP1 as reflected in the gradual increase in transition midpoint temperature, enthalpy of transition, and conformational entropy. Our studies suggest that the presence of counter ions in the molecular environment of HABP1 leads to dramatic reduction of the intramolecular electrostatic repulsion either by de-ionizing the charged amino acid residues or by direct binding leading to a more stable conformation. A regulation on cellular HA-HABP1 interaction by changes in pH and ionic strength may exist, because the more stable conformation attained at higher ionic strength or at acidic pH showed maximum affinity toward HA as probed either in solid phase binding assay on HA-immobilized plates or an in-solution binding assay using intrinsic fluorescence of HABP1.