RESUMO

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that is a disease-modifying drug candidate for Parkinson's disease. CDNF has pleiotropic protective effects on stressed cells, but its mechanism of action remains incompletely understood. Here, we use state-of-the-art advanced structural techniques to resolve the structural basis of CDNF interaction with GRP78, the master regulator of the unfolded protein response (UPR) pathway. Subsequent binding studies confirm the obtained structural model of the complex, eventually revealing the interaction site of CDNF and GRP78. Finally, mutating the key residues of CDNF mediating its interaction with GRP78 not only results in impaired binding of CDNF but also abolishes the neuroprotective activity of CDNF-derived peptides in mesencephalic neuron cultures. These results suggest that the molecular interaction with GRP78 mediates the neuroprotective actions of CDNF and provide a structural basis for development of next generation CDNF-based therapeutic compounds against neurodegenerative diseases.

Assuntos

Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Resposta a Proteínas não Dobradas , Chaperona BiP do Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Animais , Ligação Proteica , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Neurônios/metabolismo , Modelos Moleculares , Sítios de Ligação

RESUMO

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.

Assuntos

Integrina beta3/metabolismo , Talina/química , Talina/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Humanos , Integrina beta3/química , Leucina/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutagênese , Polilisina/química , Domínios Proteicos , Dobramento de Proteína , Talina/genética

RESUMO

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.

Assuntos

Integrina beta3 , Talina , Adesão Celular , Análise por Conglomerados , Integrina beta3/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Talina/genética , Talina/metabolismo

RESUMO

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C's cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

Assuntos

Calpaína/metabolismo , Proteínas do Capsídeo/metabolismo , Infecções por Enterovirus/virologia , Enterovirus/metabolismo , Poliproteínas/metabolismo , Animais , Capsídeo/metabolismo , Células Cultivadas , Glicoproteínas/farmacologia , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Proteólise , Ratos , Proteínas Virais/metabolismo

RESUMO

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

Assuntos

Avidina/metabolismo , Biotina/metabolismo , Progesterona/sangue , Progesterona/metabolismo , Animais , Avidina/química , Sítios de Ligação , Bioensaio , Biotina/química , Cães , Ligantes , Modelos Moleculares , Progesterona/química , Ligação Proteica

RESUMO

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

Assuntos

Citocinas/metabolismo , Lectinas/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Códon sem Sentido/genética , Citocinas/genética , Dexametasona/farmacologia , Resistência à Doença/imunologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Lectinas/genética , Depleção Linfocítica , Morfolinos/farmacologia , Mutação/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/efeitos dos fármacos , Análise de Sobrevida , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética

RESUMO

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2Apro and 3Cpro), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2Apro cause direct damage to the infected heart and tools to investigate 2Apro and 3Cpro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2Apro and 3Cpro; Two monoclonal 2Apro antibodies and one 3Cpro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3Cpro antibody detects all of the EV species B (EV-B) viruses tested and that the 2Apro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2Apro and 3Cpro, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.

Assuntos

Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Cisteína Endopeptidases/imunologia , Enterovirus Humano B/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Proteínas Virais/imunologia , Proteases Virais 3C , Animais , Antígenos Virais/genética , Células Cultivadas , Clonagem Molecular , Cisteína Endopeptidases/genética , Enterovirus Humano B/enzimologia , Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Sorogrupo , Proteínas Virais/genética

RESUMO

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.

Assuntos

Infecções por Coxsackievirus/prevenção & controle , Enterovirus Humano A/imunologia , Imunogenicidade da Vacina , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Infecções por Coxsackievirus/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/isolamento & purificação , Feminino , Formaldeído/farmacologia , Camundongos , Polissorbatos/farmacologia , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/isolamento & purificação , Cultura de Vírus

RESUMO

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag") act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

Assuntos

Biotina/química , Proteínas de Transporte/química , Marcadores de Afinidade , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica

RESUMO

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.

Assuntos

Anticorpos Monoclonais/administração & dosagem , Autoanticorpos/biossíntese , Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/imunologia , Hospedeiro Imunocomprometido , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Transglutaminases/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Doença Celíaca/genética , Doença Celíaca/patologia , Cricetulus , Feminino , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Glutens/química , Glutens/imunologia , Humanos , Imunoglobulina A/biossíntese , Imuno-Histoquímica , Inflamação , Injeções Intraperitoneais , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Camundongos Nus , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transglutaminases/genética

RESUMO

UNLABELLED: Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid. IMPORTANCE: The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.

Assuntos

Bacteriófagos/metabolismo , Proteínas do Capsídeo/metabolismo , Lipídeos/química , Thermus/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Bacteriófagos/química , Bacteriófagos/genética , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Metabolismo dos Lipídeos , Modelos Moleculares , Dados de Sequência Molecular , Eletricidade Estática , Thermus/química , Thermus/virologia , Vírion/química , Vírion/genética , Vírion/metabolismo

RESUMO

Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.

Assuntos

Avidina/química , Avidina/metabolismo , Técnicas Biossensoriais , Biotina/química , Células 3T3 , Animais , Avidina/genética , Sítios de Ligação , Biotinilação , Varredura Diferencial de Calorimetria , Galinhas , Camundongos , Mutação , Ressonância de Plasmônio de Superfície

RESUMO

Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin-bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin-free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability. Conventional triple resonance experiments for fully protonated proteins combined with methyl-detection optimized experiments acquired at 58°C were adequate for the structure determination of this 56 kDa protein. The model-free parameters derived from the 15N relaxation data reveal a remarkably rigid protein at 58°C in both the biotin-bound and the free forms. The H/D exchange experiments indicate a notable increase in hydrogen protection upon biotin binding.

Assuntos

Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Modelos Moleculares , Peso Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Termodinâmica

RESUMO

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.

Assuntos

Avidina/química , Proteínas de Peixes/química , Genoma , Glicoproteínas/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Avidina/genética , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Cristalografia por Raios X , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Brânquias/embriologia , Brânquias/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Gônadas/embriologia , Gônadas/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo

RESUMO

Avidin is a homotetrameric ~56 kDa protein found in chicken egg white. Avidin's ability to bind biotin with a very high affinity has widely been exploited in biotechnological applications. Protein engineering has further diversified avidin's feasibility. ChiAVD(I117Y) is a product of rational protein engineering. It is a hyperthermostable synthetic hybrid of avidin and avidin-related protein 4 (AVR4). In this chimeric protein a 23-residue segment in avidin has been replaced with the corresponding sequence found in AVR4, and a point mutation at subunit interface 1-3 (and 2-4) has been introduced. Here we report the backbone and sidechain resonance assignments of the biotin-bound form of ChiAVD(I117Y) as well as the backbone resonance assignments of the free form.

Assuntos

Avidina/química , Avidina/metabolismo , Biotina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ligantes , Peso Molecular , Ligação Proteica

RESUMO

Bradavidin is a homotetrameric biotin-binding protein from Bradyrhizobium japonicum, a nitrogen fixing and root nodule-forming symbiotic bacterium of the soybean. Wild-type (wt) bradavidin has 138 amino acid residues, whereas the C-terminally truncated core-bradavidin has only 118 residues. We have solved the X-ray structure of wt bradavidin and found that the C-terminal amino acids of each subunit were uniquely bound to the biotin-binding pocket of an adjacent subunit. The biotin-binding pocket occupying peptide (SEKLSNTK) was named "Brad-tag" and it serves as an intrinsic stabilizing ligand in wt bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and a binding affinity of ∼25 µM was measured. In order to study the potential of Brad-tag, a green fluorescent protein tagged with Brad-tag was prepared and successfully concentrated from a bacterial cell lysate using core-bradavidin-functionalized Sepharose resin.

Assuntos

Bradyrhizobium , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biotina/metabolismo , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato

RESUMO

Kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen (PSA), is the most useful biomarker for prostate cancer (PCa). KLK3 is suggested to play a role in regulating cancer growth through anti-angiogenic activity in vivo and in vitro. This feature, together with its specificity for prostate tissue, makes KLK3 an intriguing target for the design of new therapies for PCa. 3D pharmacophores for KLK3-stimulating compounds were generated based on peptides that bind specifically to KLK3 and increase its enzymatic activity. As a result of pharmacophore-based virtual screening, four small, drug-like compounds with affinity for KLK3 were discovered and validated by capillary differential scanning calorimetry. One of the compounds also stimulated the activity of KLK3, and is therefore the first published small molecule with such an activity.

Assuntos

Calicreínas/química , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Varredura Diferencial de Calorimetria , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Humanos , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Antígeno Prostático Específico , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/farmacologia

RESUMO

A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films.

Assuntos

Avidina/metabolismo , Materiais Biocompatíveis/síntese química , Biotina/metabolismo , Géis/química , Proteínas Imobilizadas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Avidina/genética , Materiais Biocompatíveis/metabolismo , Biotina/química , Cromatografia de Afinidade , Clonagem Molecular , Reagentes de Ligações Cruzadas/química , Cisteína/genética , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Géis/metabolismo , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Tinta , Transição de Fase , Plasmídeos , Reação em Cadeia da Polimerase , Polímeros/química , Polímeros/metabolismo , Propilaminas , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Silanos/química , Succinimidas/química , Propriedades de Superfície

RESUMO

BACKGROUND: Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. RESULTS: Avidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 µM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between ß-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins. CONCLUSIONS: The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.

Assuntos

Avidina/química , Testosterona/metabolismo , Avidina/genética , Avidina/metabolismo , Sítios de Ligação , Varredura Diferencial de Calorimetria , Biblioteca Gênica , Cinética , Ligantes , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Testosterona/química

RESUMO

A stable, bioactive cellulose acetate (CA) surface was developed by functionalizing the surface with highly thermostable avidin form. The CA films were first functionalized with a mixture of 3-aminopropyltrimethoxysilane and tetraethoxysilane to introduce free amino groups onto the surface of CA films. Free amino groups were functionalized with glutaraldehyde to obtain an activated surface for covalent biomolecule immobilization. A genetically engineered, high-affinity biotin-binding protein chimeric avidin, ChiAVD(I117Y), was used for biofunctionalization of the surface. The chimeric avidin protein has an increased stability in chemically harsh conditions and at high temperature when compared to wt (strept)avidin. The biological activity, i.e., biotin-binding capacity, of the immobilized protein was probed by [(3)H]-biotin. The activity of the chimeric avidin on functionalized CA films was fully retained over the three months' study period. The biotin-binding capacity of the immobilized chimeric avidin was compared to that of immobilized streptavidin, chicken avidin, and rhizavidin and significant differences between proteins were detected. The developed material offers a valuable platform for the development of inexpensive in vitro diagnostics and also supports biosensing applications that are required to operate under demanding conditions.

Assuntos

Avidina/química , Celulose/análogos & derivados , Proteínas Recombinantes de Fusão/química , Celulose/química , Microscopia Eletrônica de Varredura , Temperatura