RESUMO
Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Zinco/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Proteínas Intrinsicamente Desordenadas/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/síntese química , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Timosina/química , Timosina/metabolismoRESUMO
INTRODUCTION: A national consensus panel was convened to develop recommendations on future directions for home-based dementia care (HBDC). METHODS: The panel summarized advantages and challenges of shifting to HBDC as the nexus of care and developed consensus-based recommendations. RESULTS: The panel developed five core recommendations: (1) HBDC should be considered the nexus of new dementia models, from diagnosis to end of life in dementia; (2) new payment models are needed to support HBDC and reward integration of care; (3) a diverse new workforce that spans the care continuum should be prepared urgently; (4) new technologies to promote communication, monitoring/safety, and symptoms management must be tested, integrated, and deployed; and (5) targeted dissemination efforts for HBDC must be employed. DISCUSSION: HBDC represents a promising paradigm shift to improve care for those living with dementia and their family caregivers: these recommendations provide a framework to chart a course forward for HBDC.
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Consenso , Efeitos Psicossociais da Doença , Demência/enfermagem , Demência/psicologia , Serviços de Assistência Domiciliar , Cuidadores/psicologia , Cuidadores/estatística & dados numéricos , Análise Custo-Benefício/métodos , Análise Custo-Benefício/estatística & dados numéricos , Demência/diagnóstico , HumanosRESUMO
Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/síntese química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fases de Leitura Aberta , Estereoisomerismo , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The number and size of tiger populations continue to decline owing to habitat loss, habitat fragmentation and poaching of tigers and their prey. As a result, tiger populations have become small and highly structured. Current populations have been isolated since the early 1970s or for approximately seven generations. The objective of this study is to explore how inbreeding may be affecting the persistence of remaining tiger populations and how dispersal, either natural or artificial, may reduce the potentially detrimental effect of inbreeding depression. We developed a tiger simulation model and used published levels of genetic load in mammals to simulate inbreeding depression. Following a 50 year period of population isolation, we introduced one to four dispersing male tigers per generation to explore how gene flow from nearby populations may reduce the negative impact of inbreeding depression. For the smallest populations, even four dispersing male tigers per generation did not increase population viability, and the likelihood of extinction is more than 90% within 30 years. Unless habitat connectivity is restored or animals are artificially introduced in the next 70 years, medium size wild populations are also likely to go extinct, with only four to five of the largest wild tiger populations likely to remain extant in this same period without intervention. To reduce the risk of local extinction, habitat connectivity must be pursued concurrently with efforts to increase population size (e.g. enhance habitat quality, increase habitat availability). It is critical that infrastructure development, dam construction and other similar projects are planned appropriately so that they do not erode the extent or quality of habitat for these populations so that they can truly serve as future source populations.
Assuntos
Fluxo Gênico , Genética Populacional , Endogamia , Tigres/genética , Animais , Conservação dos Recursos Naturais , Ecossistema , Feminino , Masculino , Modelos Genéticos , Nepal , Densidade DemográficaRESUMO
CsSnI(3) is an unusual perovskite that undergoes complex displacive and reconstructive phase transitions and exhibits near-infrared emission at room temperature. Experimental and theoretical studies of CsSnI(3) have been limited by the lack of detailed crystal structure characterization and chemical instability. Here we describe the synthesis of pure polymorphic crystals, the preparation of large crack-/bubble-free ingots, the refined single-crystal structures, and temperature-dependent charge transport and optical properties of CsSnI(3), coupled with ab initio first-principles density functional theory (DFT) calculations. In situ temperature-dependent single-crystal and synchrotron powder X-ray diffraction studies reveal the origin of polymorphous phase transitions of CsSnI(3). The black orthorhombic form of CsSnI(3) demonstrates one of the largest volumetric thermal expansion coefficients for inorganic solids. Electrical conductivity, Hall effect, and thermopower measurements on it show p-type metallic behavior with low carrier density, despite the optical band gap of 1.3 eV. Hall effect measurements of the black orthorhombic perovskite phase of CsSnI(3) indicate that it is a p-type direct band gap semiconductor with carrier concentration at room temperature of â¼ 10(17) cm(-3) and a hole mobility of â¼585 cm(2) V(-1) s(-1). The hole mobility is one of the highest observed among p-type semiconductors with comparable band gaps. Its powders exhibit a strong room-temperature near-IR emission spectrum at 950 nm. Remarkably, the values of the electrical conductivity and photoluminescence intensity increase with heat treatment. The DFT calculations show that the screened-exchange local density approximation-derived band gap agrees well with the experimentally measured band gap. Calculations of the formation energy of defects strongly suggest that the electrical and light emission properties possibly result from Sn defects in the crystal structure, which arise intrinsically. Thus, although stoichiometric CsSnI(3) is a semiconductor, the material is prone to intrinsic defects associated with Sn vacancies. This creates highly mobile holes which cause the materials to appear metallic.
RESUMO
Metal ions have been shown to play a critical role in ß-amyloid (Aß) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aß complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aß complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aß16, N-acetyl-Aß16, Aß28, N-acetyl-Aß28, and Aß28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aß16 was found to be 1.1 (±0.13) × 10(9) and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aß28 variants lacking a potential Cu(2+)-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu(2+) binding to Aß and other intrinsically disordered peptides.
Assuntos
Peptídeos beta-Amiloides/química , Cobre/química , Termodinâmica , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Calorimetria , Cobre/metabolismoRESUMO
With a simple tandem iterated sequence, the carboxyl terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II) serves as the central coordinator of mRNA synthesis by harmonizing a diversity of sequential interactions with transcription and processing factors. Despite intense research interest, many key questions regarding functional and evolutionary constraints on the CTD remain unanswered; for example, what selects for the canonical heptad sequence, its tandem array across organismal diversity, and constant CTD length within given species and finally and how a sequence-identical, repetitive structure can orchestrate a diversity of simultaneous and sequential, stage-dependent interactions with both modifying enzymes and binding partners? Here we examine comparative sequence evolution of 58 RNAP II CTDs from diverse taxa representing all six major eukaryotic supergroups and employ integrated evolutionary genetic, biochemical, and biophysical analyses of the yeast CTD to further clarify how this repetitive sequence must be organized for optimal RNAP II function. We find that the CTD is composed of indivisible and independent functional units that span diheptapeptides and not only a flexible conformation around each unit but also an elastic overall structure is required. More remarkably, optimal CTD function always is achieved at approximately wild-type CTD length rather than number of functional units, regardless of the characteristics of the sequence present. Our combined observations lead us to advance an updated CTD working model, in which functional, and therefore, evolutionary constraints require a flexible CTD conformation determined by the CTD sequence and tandem register to accommodate the diversity of CTD-protein interactions and a specific CTD length rather than number of functional units to correctly order and organize global CTD-protein interactions. Patterns of conservation of these features across evolutionary diversity have important implications for comparative RNAP II function in eukaryotes and can more clearly direct specific research on CTD function in currently understudied organisms.
Assuntos
Sequência Conservada/genética , Evolução Molecular , RNA Polimerase II/química , RNA Polimerase II/genética , Sequência de Aminoácidos , Dicroísmo Circular , Variação Genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Fenótipo , Fosforilação , Maleabilidade , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
New knee brace designs are available that have the potential to improve patient outcomes relative to traditional bracing. For the indications of post-knee injury/surgery recovery, conservative management of knee osteoarthritis (OA), total knee arthroplasty (TKA) pre-habilitation, and the treatment of post-surgical extension deficits/flexion contractures, innovative new bracing designs merit review and discussion. The researchers requested information from industry brace manufacturers, and from the information received, have selected those products considered significant improvements over traditional functional brace designs for review in this article. Clinical research supporting the benefits of the innovative products listed in the article have been cited when available. The authors are both Certified Orthotists with over 50 years of combined knee bracing experience.
RESUMO
Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.
Assuntos
Proteínas de Insetos/química , Seda/química , Dicroísmo Circular , Elasticidade , Conformação Proteica , TemperaturaRESUMO
OBJECTIVES: The authors intended to investigate if 28-mm cryoballoon (CB) ablation also modifies the 4 major atrial ganglionaated plexi (GP). BACKGROUND: The major atrial GP facilitate the initiation and maintenance of atrial fibrillation (AF). The 28-mm CB covers a large surface area of the left atrium and probably the GP areas. METHODS: High-frequency stimulation (20 Hz) was delivered to the area of anterior right GP (ARGP), inferior right GP, superior left (SLGP), and inferior left GP (ILGP). Positive GP sites were defined as a prolongation of R-wave to R-wave (RR) interval during AF by >50%. The area of each GP before and after CB ablation was compared. RESULTS: A total of 18 patients with paroxysmal AF who underwent CB and radiofrequency ablation and had positive GP sites were reviewed. The Wilcoxon signed-rank test was used to assess the effects of CB ablation on each GP. There was a statistically significant difference in the area of all 4 GP after CB ablation: 1) ARGP area: 2.9 cm2 (interquartile range [IQR]: 2.1 to 3.5 cm2) pre-CB, 0.1 cm2 (IQR: 0 to 0.6 cm2) post-CB, p = 0.0002; 2) inferior right GP area: 2.1 cm2 (IQR: 0.9 to 2.9 cm2) pre-CB, 0.5 cm2 (IQR: 0 to 1.7 cm2) post-CB, p = 0.001; 3) SLGP area: 1.4 cm2 (IQR: 0.6 to 2.4 cm2) pre-CB, 0 cm2 (IQR: 0 to 0 cm2) post-CB, p = 0.0002; and 4) ILGP area: 1.3 cm2 (IQR: 0.3 to 2.2 cm2) pre-CB, 0.3 cm2 (IQR: 0 to 1.6 cm2) post-CB, p = 0.008. CONCLUSIONS: The surface area of all 4 of the major atrial GP was substantially reduced by CB ablation. The SLGP and ARGP had the largest, whereas the ILGP had the least percent of reduction following CB ablation. Part of the therapeutic effects of CB ablation may result from modifying the 4 major atrial GP.
Assuntos
Vias Autônomas/cirurgia , Ablação por Cateter/métodos , Coração/inervação , Idoso , Fibrilação Atrial/cirurgia , Vias Autônomas/efeitos da radiação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
It appears that fiber-forming proteins are not an exclusive group but that, with appropriate conditions, many proteins can potentially aggregate and form fibrils; though only certain proteins, for example, amyloids and silks, do so under normal physiological conditions. Even so, this suggests a ubiquitous aggregation mechanism in which the protein environment is at least as important as the sequence. An ideal model system in which forced and natural aggregation has been observed is silk. Silks have evolved specifically to readily form insoluble ordered structures with a wide range of structural functionality. The animal, be it silkworm or spider, will produce, store, and transport high molecular weight proteins in a complex environment to eventually allow formation of silk fibers with a variety of mechanical properties. Here we review fiber formation and its prerequisites, and discuss the mechanism by which the animal facilitates and modulates silk assembly to achieve controlled protein aggregation.
Assuntos
Dobramento de Proteína , Estrutura Secundária de Proteína , Seda/química , AnimaisRESUMO
BACKGROUND: This literature review was undertaken to determine if commonly held views about the benefits of a bone bridge technique are supported by the literature. METHODS: Four databases were searched for articles pertaining to surgical strategies specific to a bone bridge technique of the transtibial amputee. A total of 35 articles were identified as potential articles. Authors included methodology that was applied to separate topics. Following identification, articles were excluded if they were determined to be low quality evidence or not pertinent. RESULTS: Nine articles were identified to be pertinent to one of the topics: Perioperative Care, Acute Care, Subjective Analysis and Function. Two articles sorted into multiple topics. Two articles were sorted into the Perioperative Care topic, 4 articles sorted into the Acute Care topic, 2 articles into the Subjective Analysis topic and 5 articles into the Function topic. DISCUSSION: There are no high quality (level one or two) clinical trials reporting comparisons of the bone bridge technique to traditional methods. There is limited evidence supporting the clinical outcomes of the bone bridge technique. There is no agreement supporting or discouraging the perioperative and acute care aspects of the bone bridge technique. There is no evidence defining an interventional comparison of the bone bridge technique. CONCLUSION: Current level III evidence supports a bone bridge technique as an equivalent option to the non-bone bridge transtibial amputation technique. Formal level I and II clinical trials will need to be considered in the future to guide clinical practice. Clinical relevance Clinical Practice Guidelines are evidence based. This systematic literature review identifies the highest quality evidence to date which reports a consensus of outcomes agreeing bone bridge is as safe and effective as alternatives. The clinical relevance is understanding bone bridge could additionally provide a mechanistic advantage for the transtibial amputee.
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Amputação Cirúrgica/métodos , Traumatismos da Perna/cirurgia , Tíbia/cirurgia , Membros Artificiais , HumanosRESUMO
The C-terminal region of EC-SOD (extracellular superoxide dismutase) mediates the binding to both heparin/heparan sulphate and type I collagen. A mutation (Arg213-->Gly; R213G) within this extracellular matrix-binding region has recently been implicated in the development of heart disease. This relatively common mutation affects the heparin affinity, and the concentration of EC-SOD in the plasma of R213G homozygous individuals is increased 10- to 30-fold. In the present study we confirm, using R213G EC-SOD purified from a homozygous individual, that the heparin affinity is reduced. Significantly, the collagen affinity of the R213G EC-SOD variant was similarly affected and both the heparin and collagen affinities were reduced by 12-fold. Structural analysis of synthetic extracellular matrix-binding regions suggests that the mutation alters the secondary structure. We conclude that the increased concentration of EC-SOD in the plasma of R213G carriers is caused by a reduction in both heparin and collagen affinities.
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Arginina/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Glicina/metabolismo , Heparina/metabolismo , Superóxido Dismutase/sangue , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Aorta/enzimologia , Arginina/química , Arginina/genética , Cromatografia de Afinidade/métodos , Cromatografia em Agarose/métodos , Dicroísmo Circular/métodos , Glicina/química , Glicina/genética , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Especificidade por Substrato , Superóxido Dismutase/química , Superóxido Dismutase/genéticaRESUMO
As a natural outgrowth of the dental professional's role in recognizing and reporting child abuse the topic has been broadened in recent years to domestic violence, that is child, spouse/intimate partner, disabled and elder abuse. Forty years ago in the US there were 662 cases of child abuse reported to authorities. Today that reported number is in excess of 3 million per year [D. Wiese, D. Daro, Current trends in reporting and fatalities; the results of the 1994 annual 50 state survey, National Committee to Prevent Child Abuse, Working Paper 808, 1995]. The "dirty secret" of spousal/intimate partner violence is believed to affect 3-4 million individuals per year in the US. Studies have also found that between 50 and 70% of these perpetrators also abuse their children or those of their intimate partner [J. Kessman, Domestic violence, identifying the deadly silence, Texas Dent. J. (2000) 43]. Just as child abuse is most often manifested in the head or neck regions, likewise the evidence of physical violence to intimate partners and the elderly can be seen in the head or neck regions. The insidious part of partner and elder abuse is that often the largest component of these behaviors is psychological, emotional and indirect neglect, which leave no physical evidence [M. Bowers, Forensic Dental Evidence: An Investigator's Handbook, Elesevier, San Diego, CA, 2004, p. 119].
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Violência Doméstica , Odontologia Legal , Programas de Rastreamento/métodos , Idoso , Criança , Pessoas com Deficiência , HumanosRESUMO
The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and a CCD spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, fast sample freezing, fast alignment of the sample, accurate and stable sample temperatures, and small size and portability of the Dewar/cryostat cryogenic unit. When coupled with the fast data acquisition rates of the CCD spectrograph, this Dewar/cryostat is capable of supporting cryogenic luminescence spectroscopic measurements on luminescent samples at a series of known, stable temperatures in the 77-300 K range. A temperature-dependent study of the oxygen quenching of luminescence in a rhodium(III) transition metal complex is presented as an example of the type of investigation possible with this Dewar/cryostat. In the context of this apparatus, a stable temperature for cryogenic spectroscopy means a luminescent sample that is thermally equilibrated with either liquid nitrogen or gaseous nitrogen at a known measureable temperature that does not vary (ΔT < 0.1 K) during the short time scale (~1-10 sec) of the spectroscopic measurement by the CCD. The Dewar/cryostat works by taking advantage of the positive thermal gradient dT/dh that develops above liquid nitrogen level in the Dewar where h is the height of the sample above the liquid nitrogen level. The slow evaporation of the liquid nitrogen results in a slow increase in h over several hours and a consequent slow increase in the sample temperature T over this time period. A quickly acquired luminescence spectrum effectively catches the sample at a constant, thermally equilibrated temperature.
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Congelamento , Medições Luminescentes/métodos , Análise Espectral , Temperatura Baixa , NitrogênioRESUMO
Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.
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Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Calibragem , Dicroísmo Circular , Humanos , Espectrometria de Massas , Padrões de Referência , Ressonância de Plasmônio de SuperfícieRESUMO
The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.
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Proteínas de Bactérias/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Temperatura Baixa , Escherichia coli/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos , Fator de Iniciação 2 em Procariotos/genética , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por RestriçãoRESUMO
Spider silk is spun in a complex and unique process, thought to depend on a hydrophobic conversion of a predominantly disordered to a beta-sheet rich protein structures. To test this hypothesis we monitored the effect of cationic (DOTAC) and anionic (alkyl sulfate) detergents and of (ii) solvent polarity using a series of alcohols on the secondary structure transition in dilute solutions of native spidroin. Our results showed that the detergents hydrophilic head charge and hydrophobic tail length cooperatively induced either a transition to the beta-sheet rich form or a stable helical state. Changing the solvent polarity showed that HFIP and TFE induced formation of stable helical forms whereas MeOH, EtOH and IsoP induced a kinetically driven formation of beta-sheet rich structure.
Assuntos
Seda/química , 2-Propanol/química , Álcoois/química , Animais , Ânions , Sítios de Ligação , Cátions , Dicroísmo Circular , Detergentes/química , Detergentes/farmacologia , Etanol/química , Fibroínas/química , Proteínas de Insetos/química , Cinética , Metanol/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Compostos de Amônio Quaternário/química , Solventes/química , Aranhas , Sulfatos/farmacologia , Termodinâmica , Fatores de TempoRESUMO
The complexes M(3)[Pt(SnX(3))(5)] (M = Bu(4)N(+), PhCH(2)PPh(3)(+); X = Cl, Br), cis-M(2)[PtX(2)(SnX(3))(2)] (M = Bu(4)N(+), PhCH(2)PPh(3)(+), CH(3)PPh(3)(+), Pr(4)N(+); X = Cl, Br), and [PhCH(2)PPh(3)](2)[PtBr(3)(SnBr(3))] have been prepared and characterized by (119)Sn and (195)Pt NMR, far-infrared, and electronic absorption and emission spectroscopies. In acetone solutions the [Pt(SnX(3))(5)](3)(-) ions retain their trigonal bipyramidal structures but are stereochemically nonrigid as evidenced by (119)Sn and (195)Pt NMR spectroscopy. For [Pt(SnCl(3))(5)](3)(-) spin correlation is preserved between 183 and 363 K establishing that the nonrigidity is due to intramolecular tin site exchange, probably via Berry pseudorotation. Whereas, [Pt(SnCl(3))(5)](3)(-) does not undergo loss of SnCl(3)(-) or SnCl(2) to form either [Pt(SnCl(3))(4)](2)(-) or [PtCl(2)(SnCl(3))(2)](2)(-), [Pt(SnBr(3))(5)](3)(-) is not stable in acetone solution in the absence of excess SnBr(2) and forms [PtBr(2)(SnBr(3))(2)](2)(-) and [PtBr(3)(SnBr(3))](2)(-) by loss of SnBr(2). Similarly, [PtCl(2)(SnCl(3))(2)](2)(-) is stable in acetone at ambient temperatures but disproportionates at elevated temperatures and [PtBr(2)(SnBr(3))(2)](2)(-) loses SnBr(2) in acetone to form [PtBr(3)(SnBr(3))](2)(-). The crystal structures of methyltriphenylphosphonium cis-dibromobis(tribromostannyl)platinate(II) and benzyltriphenylphosphonium tribromo(tribromostannyl)platinate(II) have been determined. Both compounds crystallize in the triclinic space group P&onemacr; in unit cells with a = 12.293(16) Å, b = 12.868(6) Å, c = 25.047(8) Å, alpha = 96.11(3) degrees, beta = 91.06(3) degrees, gamma = 116.53(3) degrees, rho(calc) = 2.30 g cm(-)(3), Z = 3 and with a = 11.046(7) Å, b = 14.164(9) Å, c = 22.549(10) Å, alpha = 89.44(4) degrees, beta = 83.32(5) degrees, gamma = 68.31(5) degrees, rho(calc) = 1.893 g cm(-)(3), Z = 2, respectively. Least-squares refinements converged at R = 0.057 and 0.099 for 4048 and 4666 independent observed reflections with I/sigma(I) > 3.0 and I/sigma(I) > 2.0, respectively. For the former, the asymmetric unit contains 1.5 cis-[PtBr(2)(SnBr(3))(2)](2)(-) ions, 0.5 of which is disordered in such a way as to be pseudocentrosymmetric. This disordering involves a half-occupied PtBr(2) unit appearing on either side of the center. Simultaneously, one bromine from each SnBr(3) ligand changes sides while the other two bromines appear in average positions with very small displacements between their positions. The Pt-Sn distance in [PtBr(3)(SnBr(3))](2)(-) (2.486(3) Å) is slightly shorter than that incis-[PtBr(2)(SnBr(3))(2)](2)(-) (2.4955(3) Å, average), and both are significantly longer than that previously found in cis-[PtCl(2)(SnCl(3))(2)](2)(-) (2.3556 Å, average), which is not consistent with the relative magnitudes of the (1)J((195)Pt-(119)Sn) coupling constants (28 487, 25 720, and 27 627 Hz, respectively). From our electronic absorption and emission studies of the Pt-SnX(3)(-) complexes, we conclude that (a) the low-energy transitions are d-d transitions analogous to those found in [PtX(4)](2)(-) systems, (b) the SnCl(3)(-) ligand is a stronger sigma donor than SnBr(3)(-), (c) the triplet state from which the emission occurs is split by spin-orbit coupling into different spin-orbit states, (d) a forbidden spin-orbit state must lie at or near the bottom of the spin-orbit manifold, (e) the solid state crystal environment perturbs the platinum-tin halide electronic states, and (f) dispersion of the samples in solvents changes this perturbation, which can be rationalized in terms of an in-plane distortion of the square planar platinum coordination sphere.