RESUMO
The surfaces of the polyacrylamide cryogels were coated with L-tryptophan (cryogel-Trp) or L-phenylalanine (cryogel-Phe) to enhance crude leaf extract-derived ora-pro-nobis (OPN) protein binding via pseudo-specific hydrophobic interactions. Cryogels functionalized with amino acids were prepared and characterized through morphological, hydrodynamic, and thermal analyses. The adsorption capacities of cryogel-Phe and cryogel-Trp were evaluated in terms of type (sodium sulfate or sodium phosphate) and concentration (0.02 or 0.10â¯molâL-1) of saline solution, pH (4.0, 5.5, or 7.0), and NaCl concentration (0.0 or 0.5â¯molâL-1). The cryogel-Phe presented a higher adsorptive capacity, achieving its maximum value (qâ¯=â¯92.53â¯mgâg-1) when the crude OPN crude leaf extract was diluted in sodium sulfate 0.02â¯molâL-1â¯+â¯NaCl 0.50â¯molâL-1, at pHâ¯=â¯7.0. The dilution rate significantly (pâ¯<â¯0.05) affected the recovered protein amount after the adsorption and elution processes, reaching 94.45% when the feedstock solution was prepared with a crude extract 5 times. The zeta potential for the eluted OPN proteins was 5.76â¯mV (pHâ¯=â¯3.23) for both dilution rates. The secondary structure composition mainly included ß-sheets (46.50%) and α-helices (13.93%). The cryogel-Phe exhibited interconnected pores ranging 20-300⯵m in size, with a Young modulus of 1.51â¯MPa, and thermal degradation started at 230⯰C. These results indicate that the cryogel-Phe exhibited satisfactory properties as promising chromatography support for use in high-throughput purification of crude leaf extract-derived OPN proteins.
Assuntos
Aminoácidos Aromáticos/química , Cactaceae/química , Cromatografia de Afinidade/métodos , Criogéis/química , Proteínas de Plantas/isolamento & purificação , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Extratos Vegetais/química , Folhas de Planta/química , Proteínas de Plantas/análiseRESUMO
Phosphoinositides are phosphatidylinositol derived, well known to be second messengers in various cell signaling pathways as well as in processes such as cell differentiation, cellular stress response, gene transcription, and chromatin remodeling. The pleckstrin homology domain of phospholipase C-delta 1 is responsible for recognizing and binding to PI(4,5)P2 and for this reason has been widely used to study this phosphoinositide as a biosensor when it is conjugated to a fluorescent tag. In this work, we modified the primary structure of pleckstrin homology domain by site-specific mutagenesis to change the specificity for phosphoinositides. We obtained 3 mutants: K30A, W36F, and W36Y with different specificity to phosphoinositides. Mutant domain K30A recognized PI(4,5)P2 , PI(3,4,5)P3 , phosphatidic acid (PA), and weakly PI(3,5)P2 . Mutant domain W36F recognized all the phosphoinositides studied and the PA. Finally, mutant domain W36Y seemed to interact with PA and all the other phosphoinositides studied, except PI(3)P. The changes in recognition argue against a simple charge and nonpolar region model for these interactions and more in favor of a specific docking region with a specific recognition site. We conducted in silico modeling that explains the mechanisms behind the observed changes and showed that aromatic amino acids appear to play more important role, than previously thought, in the specificity of phospholipids' binding domains.
Assuntos
Aminoácidos Aromáticos/química , Domínios de Homologia à Plecstrina , Sequência de Aminoácidos , Animais , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipase C delta/química , RatosRESUMO
The outer membrane lipoprotein A (OmlA) belongs to a family of bacterial small lipoproteins widely distributed across the beta and gamma proteobacteria. Although the role of numerous bacterial lipoproteins is known, the biological function of OmlA remains elusive. We found that in the citrus canker pathogen, Xanthomonas axonopodis pv. citri (X. citri), OmlA is coregulated with the ferric uptake regulator (Fur) and their expression is enhanced when X. citri is grown on citrus leaves, suggesting that these proteins are involved in plant-pathogen interaction. To gain insights into the function of OmlA, its conformational and dynamic features were determined by nuclear magnetic resonance. The protein has highly flexible N- and C- termini and a structurally well defined core composed of three beta-strands and two small alpha-helices, which pack against each other forming a two-layer alpha/beta scaffold. This protein fold resembles the domains of the beta-lactamase inhibitory protein BLIP, involved in protein-protein binding. In conclusion, the structure of OmlA does suggest that this protein may be implicated in protein-protein interactions required during X. citri infection.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Lipoproteínas/química , Dobramento de Proteína , Proteínas/metabolismo , Xanthomonas axonopodis/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos Aromáticos/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Dicroísmo Circular , Cisteína/química , Medição da Troca de Deutério , Escherichia coli/genética , Genes Reporter , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Conformação Proteica , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.
Assuntos
Acústica , Aminoácidos Aromáticos/química , Proteínas/química , Análise Espectral/métodos , Absorção , Conformação Proteica , Dodecilsulfato de Sódio/química , Titulometria , Raios UltravioletaRESUMO
We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.