RESUMO
We describe the qualitative and quantitative analysis of the complexation properties towards cations of a cyclic peptoid hexamer composed of alternating α- and ß-peptoid monomers, which bear exclusively chiral (S)-phenylethyl side chains (spe) that have no noticeable chelating properties. The binding of a series of monovalent and divalent cations was assessed by 1H NMR, circular dichroism, fluorescence and molecular modelling. In contrast to previous studies on cations binding by 18-membered α-cyclopeptoid hexamers, the 21-membered cyclopeptoid cP1 did not complex monovalent cations (Na+, K+, Ag+) but showed selectivity for divalent cations (Ca2+, Ba2+, Sr2+ and Mg2+). Hexacoordinated C-3 symmetrical complexes were demonstrated for divalent cations with ionic radii around 1 Å (Ca2+ and Ba2+), while 5-coordination is preferred for divalent cations with larger (Ba2+) or smaller ionic radii (Mg2+).
Assuntos
Peptoides/química , Cátions Bivalentes/química , Dicroísmo Circular , Fluorescência , Modelos Moleculares , Estrutura Molecular , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E) with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.
RESUMO
The glycosaminoglycan (GAG) family of linear sulphated polysaccharides are involved in most regulatory processes in the extracellular matrix of higher organisms. The relationship between GAG substitution pattern and activity, however, remains unclear and experimental evidence suggests that subtle conformational factors play an important role. The difficulty of modelling these complex charged molecules shifts the burden of investigation towards experimental techniques. Recent advances in complementary physical-chemical, particularly spectroscopy-based approaches are reviewed, together with methods for analysing the resulting complex data. The prospects for combining some of these approaches and fitting them into the wider context of interactions, are also discussed.
Assuntos
Glicosaminoglicanos/química , Configuração de Carboidratos , Biologia Computacional , Humanos , Análise Espectral , Estatística como AssuntoRESUMO
A novel route for the synthesis of cyclic peptides constrained by an aliphatic bridge between two C(alpha)sites, using a triply orthogonal protecting group strategy, is described. The synthesis of the orthogonally protected bis-amino acid 1, via an enantioselective route utilizing the Schöllkopf and Evans methodologies, is first described. This is then incorporated into a short, alanine-rich peptide 13, using a novel triply orthogonal protecting group strategy to couple first one, then the other, amino acid moiety in such a way that an aliphatic bridge is formed between the i and i + 4 positions. Unexpectedly, the resulting constrained peptide does not adopt a helical conformation: instead, it is shown by CD at low temperature to adopt a left-handed type II beta-turn conformation in aqueous media and a right-handed type I beta-turn conformation in TFE.
Assuntos
Aminoácidos/síntese química , Peptídeos Cíclicos/síntese química , Aminoácidos/química , Dicroísmo Circular , Biossíntese Peptídica , Peptídeos Cíclicos/química , Estrutura Secundária de ProteínaRESUMO
Familial British dementia (FBD) is a rare neurodegenerative disorder and shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss, and progressive dementia. Immunohistochemical and biochemical analysis of plaques and vascular amyloid of FBD brains revealed that a 4 kDa peptide named ABri is the main component of the highly insoluble amyloid deposits. In FBD patients, the ABri peptide is produced as a result of a point mutation in the usual stop codon of the BRI gene. This mutation produces a BRI precursor protein 11 amino acids longer than the wild-type protein. Mutant and wild-type precursor proteins both undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids in the case of the wild-type (WT) peptide. Here we demonstrate that the intramolecular disulfide bond in ABri and the C-terminal extension are required to elongate initially formed dimers to oligomers and fibrils. In contrast, the shorter WT peptide did not aggregate under the same conditions. Conformational analyses indicate that the disulfide bond and the C-terminal extension of ABri are required for the formation of beta-sheet structure. Soluble nonfibrillar ABri oligomers were observed prior to the appearance of mature fibrils. A molecular model of ABri containing three beta-strands, and two beta-hairpins annealed by a disulfide bond, has been constructed, and predicts a hydrophobic surface which is instrumental in promoting oligomerization.
Assuntos
Amiloide/metabolismo , Demência/metabolismo , Dissulfetos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Amiloide/química , Amiloide/genética , Amiloide/ultraestrutura , Sítios de Ligação/genética , Dicroísmo Circular , Corantes , Vermelho Congo/metabolismo , Demência/genética , Demência/patologia , Dimerização , Humanos , Cinética , Glicoproteínas de Membrana , Proteínas de Membrana , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/ultraestrutura , Estrutura Secundária de Proteína/genética , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Reino UnidoRESUMO
Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.
Assuntos
Transporte Axonal , Gangliosídeos/metabolismo , Neurônios Motores/metabolismo , Fragmentos de Peptídeos/metabolismo , Toxina Tetânica/metabolismo , Animais , Bovinos , Células Cultivadas , Histidina/genética , Histidina/metabolismo , Camundongos , Neurônios Motores/citologia , Mutagênese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Ratos , Toxina Tetânica/química , Toxina Tetânica/genéticaRESUMO
The high molecular weight (HMW) subunit group of wheat seed storage proteins impart elasticity to wheat doughs and glutens. They consist of three domains: non-repetitive N- and C-terminal domains, which contain cysteine residues for covalent cross-linking, and a central domain consisting of repeated sequences. The circular dichroism and infrared (IR) spectra of an intact HMW subunit were compared with those of a peptide corresponding to the central repetitive domain expressed in Escherichia coli. This allowed the structure of the central domain to be studied in the absence of the N- and C-terminal domains and the contributions of these domains to the structure of the whole protein to be determined. In solution the peptide showed the presence of beta-turns and polyproline II-like structure. Variable temperature studies indicated an equilibrium between these two structures, the polyproline II conformation predominating at low temperatures and the beta-turn conformation at higher temperatures. IR in the hydrated solid state also indicated the presence of beta-turns and intermolecular beta-sheet structures. In contrast, spectroscopy of the whole subunit showed the presence of alpha-helix in the N- and C-terminal domains. The content of beta-sheet was also higher in the whole subunit, indicating that the N- and C-terminal domains may promote the formation of intermolecular beta-sheet structures between the repetitive sequences, perhaps by aligning the molecules to promote interaction.
Assuntos
Peptídeos/química , Proteínas de Plantas/química , Triticum/química , Sequência de Aminoácidos , Dicroísmo Circular , Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequências Repetitivas de Aminoácidos , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Reagentes de Ligações Cruzadas/farmacologia , DNA Girase , DNA Topoisomerases Tipo II/metabolismo , Dimerização , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/genética , Cinética , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Proteínas MutL , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Temperatura , Fatores de TempoRESUMO
A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer's disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Hemorragia Cerebral/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Apoptose , Biotinilação , Cromatografia em Gel , Dicroísmo Circular , Dimerização , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica , Modelos Moleculares , Mutação , Fenótipo , Conformação Proteica , Fatores de Tempo , Células Tumorais CultivadasRESUMO
H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/química , Oligopeptídeos/química , Salmonella typhimurium/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de ProteínaRESUMO
The AmiC protein in Pseudomonas aeruginosa is the negative regulator and ligand receptor for an amide-inducible aliphatic amidase operon. In the wild-type PAC1 strain, amidase expression is induced by acetamide or lactamide, but not by butyramide. A mutant strain of P. aeruginosa, PAC181, was selected for its sensitivity to induction by butyramide. The molecular basis for the butyramide inducible phenotype of P.aeruginosa PAC181 has now been determined, and results from a Thr-->Asn mutation at position 106 in PAC181-AmiC. In the wild-type PAC1-AmiC protein this residue forms part of the side wall of the amide-binding pocket but does not interact with the acetamide ligand directly. In the crystal structure of PAC181-AmiC complexed with butyramide, the Thr-->Asn mutation increases the size of the ligand binding site such that the mutant protein is able to close into its 'on' configuration even in the presence of butyramide. Although the mutation allows butyramide to be recognized as an inducer of amidase expression, the mutation is structurally sub-optimal, and produces a significant decrease in the stability of the mutant protein.
Assuntos
Adaptação Biológica/genética , Proteínas de Bactérias/genética , Proteínas Periplásmicas de Ligação , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Seleção Genética , Amidas/metabolismo , Amidas/farmacologia , Amidoidrolases/genética , Amidoidrolases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Genes Reguladores , Temperatura Alta , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Ligação Proteica , Desnaturação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.
Assuntos
Adenosina Trifosfatases/metabolismo , Ciclofilinas , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Peptidil-Prolil Isomerase F , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico , Substâncias Macromoleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Sequências Repetitivas de Aminoácidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
Non-immobilised ligand interaction assay (NILIA) by CD spectroscopy provides an excellent technique to study molecular interactions in solution. Here are discussed molecular interactions of several systems that involve hosts and ligands with wide range of molecular sizes. Cytokine rhGM-CSF (14.6 kDa) bound to alpha-chain hGM-CSF receptor fragment (2 kDa, Kd = 35 microM), proline rich peptide (1.5 kDa) bound to fynSH3 domain (8 kDa, Kd = 28 microM), tumour imaging peptide (2 kDa) bound to mucin antigenic fragment (2 kDa, Kd = 20 microM), monoclonal antibody (150 kDa) bound to antigenic protein (120 kDa, Kd = 50 nM). Reconstitution of Cytochrome b5 (Cyt b5) from apo-Cyt b5 and hemin (Kd = 1.6 nM), correct protein folding of reconstituted porphobilinogen deaminase from apo-cofactorless form achieved using the product of the enzyme catalysis, preuroporphyrinogen, rather than porphobilinogen substrate. Competition studies of bound non-chiral drugs diclofenac and diazepam to carrier proteins such as HSA in the presence of fatty acids are few of the examples of the studies carried out by NILIA-CD spectroscopy. The CD changes in either backbone, aromatic side-chains and disulphide regions were used accordingly to screen qualitatively and quantitatively ligand binding in vitro. CD data were fitted by non-linear regression to the general equilibrium reaction of a single-binding site. NILIA-CD is fast compared to NMR, gives information on conformational changes due to interaction, avoids masking of the binding site due to immobilisation and requires no radiolabelling. NILIA-CD is thus an ideal technique for interaction, activity, screening studies.
Assuntos
Sítios de Ligação , Conformação Proteica , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Ligação Competitiva , Dicroísmo Circular , Citocromos b5/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismoRESUMO
The E. coli low molecular mass penicillin-binding proteins (PBP's) are penicillin sensitive, enzymes involved in the terminal stages of peptidoglycan biosynthesesis. These PBP's are believed to anchor to the periplasmic face of the inner membrane via C-terminal amphiphilic alpha-helices but to date the only support for this hypothesis has been obtained from theoretical analysis. In this paper, the conformational behaviour of synthetic peptides corresponding to these C-terminal anchoring domains was studied as a function of solvent, pH, sodium dodecyl sulphate micelles and phospholipid (DOPC, DOPG) vesicles using circular dichroism (CD) spectroscopy. The CD data showed that in 2,2,2-trifluoroethanol or sodium dodecylsulphate, all three peptides have the capacity to form an alpha-helical conformation but in aqueous solution or in the presence of phospholipid vesicles only those peptides corresponding to the PBP5 and PBP6 C-termini were observed to do so. A pH dependent loss of alpha-helical conformation in the peptide corresponding to the PBP5 C-terminus was found to correlate with the susceptibility of PBP5 to membrane extraction. This correlation would agree with the hypothesis that an alpha-helical conformation is required for membrane interaction of the PBP5 C-terminal region.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Hexosiltransferases , Lipossomos , Muramilpentapeptídeo Carboxipeptidase/química , Fragmentos de Peptídeos/química , Peptidil Transferases , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Peptídeos/síntese química , Peptídeos/química , Fosfatidilcolinas , FosfatidilgliceróisRESUMO
Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from four molecules of porphobilinogen (PBG). The PBG-D apoenzyme is responsible for the autocatalytic synthesis and covalent attachment of a dipyrromethane cofactor at its active site. In this paper an efficient method for the purification of Escherichia coli PBG-D apoenzyme using an affinity chromatography resin is reported. Circular dichroism (CD) spectra of apoenzyme and holoenzyme were recorded and significant differences in both the backbone and aromatic region of the spectra were observed. The differences in the spectra allowed the reconstitution of holoenzyme from purified apoenzyme with PBG and preuroporphyrinogen in solution to be monitored separately by CD. Apoenzyme incubated with preuroporhyrinogen gave a CD spectrum that was much more like the CD spectrum of holoenzyme than apoenzyme incubated with PBG. The results showed clearly that the cofactor was generated much more rapidly from preuroporphyrinogen than from PBG. Changes in the CD spectrum associated with the aromatic side-chain region, in particular the contribution assigned to phenylalanine-62, were found to correlate well with the activity of the reconstituted enzyme. Phenylalanine-62 is located in close proximity to the cofactor and acts as a sensitive probe to active-site changes. The stability of the holoenzyme and apoenzyme were compared with respect to both heat and susceptibility to proteolysis. The results were consistent with a model for the apoenzyme in which, in the absence of the cofactor, the three domains of the protein are held less rigidly together, thereby making the protein more susceptible to heat denaturation and proteolysis. The CD spectrum of the holoenzyme was found to be similar at both pH 5.1 and 7.4, suggesting that the crystal structure, determined at pH 5.1, is likely to be similar at physiological pH values.
Assuntos
Apoenzimas/metabolismo , Escherichia coli/enzimologia , Hidroximetilbilano Sintase/metabolismo , Porfobilinogênio/metabolismo , Urobilinogênio/metabolismo , Apoenzimas/efeitos dos fármacos , Apoenzimas/isolamento & purificação , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Hidroximetilbilano Sintase/efeitos dos fármacos , Porfobilinogênio/farmacologia , Tripsina , Urobilinogênio/farmacologiaAssuntos
Sintase do Porfobilinogênio/química , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Quelantes , Clonagem Molecular , Ácido Edético , Estabilidade Enzimática , Escherichia coli/enzimologia , Metais/análise , Fenantrolinas , Sintase do Porfobilinogênio/biossíntese , Sintase do Porfobilinogênio/isolamento & purificação , Desnaturação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , TermodinâmicaRESUMO
The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data. The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described. This indicates that the peptide binds as a poly(L-proline) type II helix. Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure. Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1). Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1). From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/genética , Expressão Gênica/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Software , TermodinâmicaRESUMO
Abstract: The solution structure of a 20 amino acid long peptide corresponding to the region 141-160 of the envelope protein Vp1 from foot-and-mouth disease virus (FMDV) serotype A, variant A, has been determined by a combination of NMR experiments and computer calculations. The peptide contains both the immunodominant epitope as well as the sequence (RGD) used by the virus to bind the cell receptor in the initial stages of infection. These two sites have been shown to partially overlap. One hundred and thirty-five NMR distance constraints were used to obtain a set of 11 structures by distance geometry, minimization and molecular dynamics simulations. These structures were divided into two homogeneous families based upon backbone superimposition. The first and most populated family was characterized by a backbone RMS of 1.5 +/- 0.4 A, the second by a backbone RMS of 0.8 +/- 0.2 A. The two families had similar structural features and differed mainly in the backbone angles of G149. In the larger of the two families these angles favoured the formation of a loop comprising residues 147 to 152 and stabilized by a H-bond between NH of D147 and the CO of A152. In the second family, where this bond was absent, the peptide adopted in this region the shape of an irregular helix. The C-terminal half of the peptide (152-159) was similar in both families and largely helical. Similar structural features were also found within the VRGDS sequence (144-148) which was assigned to a beta-turn type IV. The features of the two families of structures were found to be different from those of the recently published X-ray structure of the antigenic loop of a chemically modified form of FMDV. Proposals accounting for these differences are provided which take into account the dual activity of the 141-160 sequence (i.e. antibody binding and cell invasion through receptor binding).
Assuntos
Aphthovirus/química , Epitopos Imunodominantes , Fragmentos de Peptídeos/química , Receptores Virais/metabolismo , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Aphthovirus/classificação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Estrutura Secundária de Proteína , SorotipagemRESUMO
Based on the CDR3 V(H) sequence of a monoclonal antibody (ASM2) raised against epithelial cancer cells, the synthetic peptide YCAREPPTRTFAYWG (EPPT1) has been found to have an appreciable affinity (Kd = 20 microM) for the deglycosylated mucin-derived peptide antigen YVTSAPDTRPAPGST (PDTRP). The technetium-radiolabelled form of this peptide has been found to be a good tumour-imaging candidate for diagnosis of breast carcinoma. Several EPPT1 peptide analogues were synthesised. A differential biostability was obtained blocking the end groups of EPPT1. The susceptibility to proteolytic degradation was significantly decreased for the C-amidated form of EPPT1 than the N-acetylated form. Using resonant mirror biosensor technique, the EPPT1 analogues were classified as active and non-active peptides according to their PDTRP-binding properties. The binding of EPPT1 to PDTRP in free solution was also determined unambiguously by CD spectroscopy. CD spectra of both active and non-active peptides showed the presence of irregular conformations in H2) and SDS above cmc. In TFE, significant degree of ordered conformations of alpha-helix or beta-turn type were induced but did not correlate well with their binding properties. In SDS below cmc a conformational difference was observed between the active and non-active peptides. The active peptides exhibited CD spectra of aggregation of beta-strand type whilst the non-active showed CD spectra similar to those in H2O and SDS above cmc, critical micelle concentration. A good correlation between the extended conformation of beta-strand type and the binding affinity of the active peptides suggests this conformation as the binding feature of the EPPT tumour-imaging peptides. These information are vital for the design of novel EPPT analogues. Any modification to improve binding affinity must retain the ability of the peptides to adopt the extended conformation of beta-strand type.