RESUMO
Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.
Assuntos
Heme , Peptídeos , CatáliseRESUMO
Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.
RESUMO
Lab-on-a-Chip (LoC), or micro-Total Analysis Systems (µTAS), is recognized as a powerful analytical technology with high capabilities, though end-user products for protein purification are still far from being available on the market. Remarkable progress has been achieved in the separation of nucleic acids and proteins using electrophoretic microfluidic devices, while pintsize devices have been developed for protein isolation according to miniaturized chromatography principles (size, charge, affinity, etc.). In this work, we review the latest advances in the fabrication of components, detection methods and commercial implementation for the separation of biological macromolecules based on microfluidic systems, with some critical remarks on the perspectives of their future development towards standardized microfluidic systems and protocols. An outlook on the current needs and future applications is also presented.
RESUMO
Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5'-TGAAACCGGTTTCA-3', which is present in all three promoters. Crystallization of native PtxS failed, and PtxS-DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10×10â µm. The crystal belonged to the trigonal space group P3, with unit-cell parameters a=b=213.71, c=71.57â Å. Only unhandled crystals grown in capillaries of 0.1â mm inner diameter diffracted X-rays to 1.92â Å resolution.
Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Pseudomonas putida , Fatores de Transcrição/química , Soluções Tampão , Crioprotetores/química , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Regiões Operadoras Genéticas , Ligação ProteicaRESUMO
Methyl-accepting chemotaxis proteins (MCPs) are transmembrane proteins that sense changes in environmental signals, generating a chemotactic response and regulating other cellular processes. MCPs are composed of two main domains: a ligand-binding domain (LBD) and a cytosolic signalling domain (CSD). Here, the crystallization of the LBD of the chemoreceptor McpS (McpS-LBD) is reported. McpS-LBD is responsible for sensing most of the TCA-cycle intermediates in the soil bacterium Pseudomonas putida KT2440. McpS-LBD was expressed, purified and crystallized in complex with two of its natural ligands (malate and succinate). Crystals were obtained by both the counter-diffusion and the hanging-drop vapour-diffusion techniques after pre-incubation of McpS-LBD with the ligands. The crystals were isomorphous and belonged to space group C2, with two molecules per asymmetric unit. Diffraction data were collected at the ESRF synchrotron X-ray source to resolutions of 1.8 and 1.9 Å for the malate and succinate complexes, respectively.
Assuntos
Proteínas de Bactérias/química , Malatos/química , Pseudomonas putida/química , Ácido Succínico/química , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Ligantes , Malatos/metabolismo , Estrutura Terciária de Proteína , Pseudomonas putida/metabolismo , Ácido Succínico/metabolismoRESUMO
The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.
Assuntos
Hidrolases/química , Rhodococcus/enzimologia , Cristalografia por Raios X , Hidrolases/genética , Isoenzimas/química , Isoenzimas/genética , Ligantes , Modelos Moleculares , Mutação , Engenharia de Proteínas , Estrutura Terciária de ProteínaRESUMO
Mexicain is a 23.8 kDa cysteine protease from the tropical plant Jacaratia mexicana. It is isolated as the most abundant product after cation-exchange chromatography of the mix of proteases extracted from the latex of the fruit. The purified enzyme inhibited with E-64 [N-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane] was crystallized by sitting-drop vapour diffusion and the structure was solved by molecular replacement at 2.1 A resolution and refined to an R factor of 17.7% (R(free) = 23.8%). The enzyme belongs to the alpha+beta class of proteins and the structure shows the typical papain-like fold composed of two domains, the alpha-helix-rich (L) domain and the beta-barrel-like (R) domain, separated by a groove containing the active site formed by residues Cys25 and His159, one from each domain. The four monomers in the asymmetric unit show one E-64 molecule covalently bound to Cys25 in the active site and differences have been found in the placement of E-64 in each monomer.
Assuntos
Papaína/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cristalização , Cristalografia , Eletroforese em Gel de Poliacrilamida , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Homologia de Sequência de AminoácidosRESUMO
Mexicain is a 23.7 kDa papain-like cysteine protease from the tropical plant Jacaratia mexicana. Extracted as a mix of proteases from the latex of the fruit, mexicain is isolated after cation-exchange chromatography as the most abundant product. The purified product inhibited with E-64 was crystallized by sitting-drop vapour diffusion in the presence of ethanolamine. Cryoprotected crystals diffracted X-rays from a home source to 1.98 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 57.36, b = 90.45, c = 80.39 A, beta = 92.64 degrees . The asymmetric unit contains four molecules of mexicain, with a corresponding crystal volume per protein weight (V(M)) of 2.24 A(3) Da(-1) and a solvent content of 45% by volume. A molecular-replacement model has been determined and refinement is in progress.
Assuntos
Caricaceae/enzimologia , Cisteína Endopeptidases/química , Proteínas de Plantas/química , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/isolamento & purificação , Proteínas de Plantas/isolamento & purificaçãoRESUMO
Very high quality crystals of tetragonal hen egg-white lysozyme were grown in the Advanced Protein Crystallization Facility (APCF) on board the Space Shuttle using a modified free-interface diffusion (FID) reactor designed ad hoc to have a longer diffusion path. This design allows the performance of true counter-diffusion experiments. Crystals were obtained under the classical chemical conditions defined 50 y ago with NaCl as a crystallizing agent and acetate pH 4.5 as a buffer. Counter-diffusion crystallization allows a "physical" instead of chemical optimization of growth conditions: indeed, this method screens for the best supersaturation conditions in a single trial and yields crystals of very high quality. A complete diffraction data set was collected at atomic resolution from one of these crystals using synchrotron radiation at the DESY-EMBL beamlines. The overall R(merge) on intensities in the resolution range 31-0.94 A was 5.2% and the data were 98.9% complete. Refinement was carried out with the programs CNS and SHELX97 to a final crystallographic R factor of 12.26% for 72 390 reflections. A mean standard uncertainty in the atomic positions of 0.024 A was estimated from inversion of blocked least-squares matrices. 22 side chains show alternate conformations and the loop 59-75 adopts in the same crystal packing two conformations that were observed for either triclinic or tetragonal lysozyme in previous high-resolution studies. In addition to 255 water molecules, the crystallizing agent (one hexacoordinated sodium ion and five chloride anions) participates in the ordered lysozyme hydration shell.
Assuntos
Muramidase/química , Aminoácidos/química , Animais , Ânions/química , Sítios de Ligação , Cátions/química , Galinhas , Cristalização , Cristalografia por Raios X , Clara de Ovo/análise , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The rocking curve of protein crystals contains a lot of useful information concerning crystal quality, most of which is lost owing to the superimposition of spurious features appearing in these fragile materials after growth, during handling and mounting. To minimize such data spoiling, an experimental setup to perform in situ X-ray diffraction experiments during crystal growth has been designed. The setup, which includes video observation to allow the correlation of crystal shape, size and growth rate with X-ray data, has been used to assess the mosaicity of tetragonal lysozyme crystals during crystal growth. The full width at half maximum (FWHM) of diffraction peaks collected from these crystals changes during the growth process as a (directly proportional) response to the growth rates and the different development of different domain blocks. These changes in the domain distribution and FWHM with time involve a 'zonation' of the crystals, which show very different rocking curves in different parts of their volume. The rocking curves recorded in situ from growing crystals are easier to understand than those from crystals that have suffered even minor handling.