RESUMO
Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.
Assuntos
Caseínas , Muramidase , Termolisina , Muramidase/química , Muramidase/metabolismo , Termolisina/química , Termolisina/metabolismo , Caseínas/química , Glicerol/química , Água/química , Glucose/química , Difração de Nêutrons , Simulação de Dinâmica MolecularRESUMO
In this paper, we applied the molecular dynamics (MD) simulations and used thermolysin as the system to study the overall protein dynamics and side chain dihedral angles across the Arrhenius break. Simulations were performed at a high temperature of 36 °C which is above the previously observed Arrhenius break, and the lower temperature of 20 °C which is below the Arrhenius break. We observed different protein dynamics and conformational heterogeneity of side chain dihedral angles of thermolysin at the two temperatures. Our results indicated that certain regions of thermolysin have a higher level of fluctuation at lower temperature. A temperature dependent dihedral angles were also observed at the two temperatures. The changes observed in the protein indicated key areas of temperature sensitivity within the protein.Communicated by Ramaswamy H. Sarma.
Assuntos
Temperatura Alta , Simulação de Dinâmica Molecular , Termolisina/química , Conformação Proteica , TemperaturaRESUMO
The majority of biological processes are regulated by enzymes, precise control over specific enzymes could create the potential for controlling cellular processes remotely. We show that the thermophilic enzyme thermolysin can be remotely activated in 17.76 MHz radiofrequency (RF) fields when covalently attached to 6.1 nm gold coated magnetite nanoparticles. Without raising the bulk solution temperature, we observe enzyme activity as if the solution was 16 ± 2 °C warmer in RF fields-an increase in enzymatic rate of 129 ± 8%. Kinetics studies show that the activity increase of the enzyme is consistent with the induced fit of a hot enzyme with cold substrate.
Assuntos
Ouro/química , Temperatura Alta , Nanopartículas de Magnetita/química , Ondas de Rádio , Termolisina/químicaRESUMO
Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH4)2SO4, Na2SO4 and MgSO4, were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.
Assuntos
Proteínas de Bactérias , Chromohalobacter/enzimologia , Peptídeo Hidrolases , Cloreto de Sódio/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Temperatura Alta , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Termolisina/química , Termolisina/metabolismoRESUMO
Metalloproteases and their inhibitors are important in numerous fundamental biochemical phenomena and medical applications. The heterocyclic organic compound, 1,10-phenanthroline, forms a complex with transition metal ions and is a Zn2+-chelating metalloprotease inhibitor; however, the mechanism of 1,10-phenanthroline-based chelation inhibition has not been fully elucidated. This study aimed to understand the structural basis of zinc metalloproteinase inhibition by 1,10-phenanthroline. Herein, the crystal structure of thermolysin was determined in the absence and presence of 1,10-phenanthroline at 1.5 and 1.8 Å, respectively. In native thermolysin, Zn2+ at the active site is tetrahedrally coordinated by His142, His146, Glu166, and water molecule and contains three Ca2+ ions, which are involved in thermostability. In the crystal structure of 1,10-phenanthroline-treated thermolysin crystal, seven 1,10-phenanthroline molecules were observed on the surface of thermolysin. These molecules are stabilized by π- π stacking interactions with aromatic amino acids (Phe63, Tyr66, Tyr110, His216, and Try251) or between the 1,10-phenanthrolines. Moreover, interactions with Ser5 and Arg101 were also observed. In this structure, Zn2+ at the active site was completely chelated, but no large conformational changes were observed in Zn2+ coordination with amino acid residues. Ca2+ at the Ca3 site exposed to the solvent was chelated by 1,10-phenanthroline, resulting in a conformational change in the side chain of Asp56 and Gln61. Based on the surface structure, for 1,10-phenanthroline to chelate a metal, it is important that the metal is exposed on the protein surface and that there is no steric hindrance impairing 1,10-phenanthroline access by the amino acids around the metal.
Assuntos
Quelantes/química , Fenantrolinas/química , Termolisina/química , Domínio Catalítico , Íons/química , Metaloproteases/química , Metais/química , Modelos Moleculares , Solventes , Água/química , Difração de Raios X/métodos , Zinco/químicaRESUMO
The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.
Assuntos
Anoxybacillus/metabolismo , Extremófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Anoxybacillus/classificação , Anoxybacillus/isolamento & purificação , Anoxybacillus/fisiologia , Brasil , Estabilidade Enzimática , Extremófilos/classificação , Extremófilos/isolamento & purificação , Extremófilos/fisiologia , Concentração de Íons de Hidrogênio , Queratinas/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Temperatura , Termolisina/química , Termolisina/metabolismo , TermotolerânciaRESUMO
Thermolysin (TL) is an industrially important zinc endopeptidase, and the prototype of the M4 family of metallopeptidases. The catalytic function of TL and its relatives is typically assessed using chromogenic or more sensitive fluorescent peptides, with the latter substrates relying on Förster resonance energy transfer (FRET). Here, we demonstrate that a FRET-quenched heptapeptide designed on the basis of the enzyme's substrate specificity (Dabcyl-FKFLGKE-EDANS) is efficiently cleaved by TL and dispase (a TL-like protease) in between the Phe3 and Leu4 residues. The specificity constants (determined at pH 7.4 and 25 °C) for TL and dispase (3.6 × 106 M-1 s-1 and 4.6 × 106 M-1 s-1, respectively) were found to be amongst the highest documented for any TL substrate. Maximal peptide cleavage rates were achieved at pH 6.5 and a temperature of 65 °C. In view of the sensitivity of the assay, concentrations as low as 10 pM TL could be detected. Furthermore, the rate of hydrolysis of Dabcyl-FKFLGKE-EDANS was slow or immeasurable with some other unrelated metallo-, serine- and cysteine proteases, suggesting that the peptide has the potential to serve as a selective substrate for TL and TL-like proteases.
Assuntos
Proteínas de Bactérias/química , Geobacillus stearothermophilus/enzimologia , Termolisina/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Especificidade por SubstratoRESUMO
Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as "CAAX proteases" targeting prenylated substrates, including a-factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique "α-barrel" structure consisting of seven transmembrane (TM) α-helices encircling a large intramembranous cavity (~14 000 Å3 ). The catalytic zinc, coordinated via a HExxH E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long-studied class of soluble ZMPs, and as a novel cavity-containing integral membrane protein protease has been minimally explored to date. Informed by homology to well-characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc-containing "ZMP Core" module surrounded by a "ZMP Accessory" module, both capped by a TM helical "α-barrel" module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α-barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.
Assuntos
Proteínas de Membrana/química , Metaloendopeptidases/química , Neprilisina/química , Termolisina/química , Sequência de Aminoácidos , Bacillus/química , Bacillus/enzimologia , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Geobacter/química , Geobacter/enzimologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Neprilisina/genética , Neprilisina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces/química , Saccharomyces/enzimologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termolisina/genética , Termolisina/metabolismoRESUMO
Aqueous-liquid crystal (LC) interfaces offer promise as responsive interfaces at which biomolecular recognition events can be amplified into macroscopic signals. However, the design of LC interfaces that distinguish between specific and non-specific protein interactions remains an unresolved challenge. Herein, we report the synthesis of amphiphilic monomers, dimers, and trimers conjugated to sulfonamide ligands via triazole rings, their assembly at aqueous-LC interfaces, and the orientational response of LCs to the interactions of carbonic anhydrase II (CAII) and serum albumin with the oligomer-decorated LC interfaces. Of six oligomers synthesized, only dimers without amide methylation were found to assemble at aqueous interfaces of nematic 4-cyano-4'-pentylbiphenyl (5CB) to induce perpendicular LC orientations. At dimer-decorated LC interfaces, we found that concentrations of CAII less than 4 µM did not measurably perturb the LC but prevented non-specific adsorption and penetration of serum albumin into the dimer-decorated interface that otherwise triggered bright, globular LC optical domains. These experiments and others (including competitive adsorption of CAII, BSA, and lysozyme) support our hypothesis that specific binding of CAII to the dimer prevents LC anchoring transitions triggered by non-specific adsorption of serum albumin. We illustrate the utility of the approach by reporting (i) the relative activity of two small-molecule inhibitors (6-ethoxy-2-benzothiazolesulfonamide and benzenesulfonamide) of CAII to sulfonamide and (ii) proteolytic digestion of a protein (CAII) by thermolysin. Overall, the results in this paper provide new insight into the interactions of proteins at aqueous-LC interfaces and fresh ideas for either blocking non-specific interactions of proteins at surfaces or reporting specific binding events at LC interfaces in the presence of non-specific proteins.
Assuntos
Compostos de Bifenilo/química , Cristais Líquidos/química , Nitrilas/química , Polímeros/química , Proteínas/química , Sulfonamidas/química , Água/química , Adsorção , Anidrase Carbônica II/química , Etil-Éteres/química , Ligantes , Microscopia , Estrutura Molecular , Muramidase/química , Ligação Proteica , Albumina Sérica/química , Propriedades de Superfície , Termolisina/química , Triazóis/química , BenzenossulfonamidasRESUMO
A nonlinear least-squares method for refining a parametric expression describing the estimated errors of reflection intensities in serial crystallographic (SX) data is presented. This approach, which is similar to that used in the rotation method of crystallographic data collection at synchrotrons, propagates error estimates from photon-counting statistics to the merged data. Here, it is demonstrated that the application of this approach to SX data provides better SAD phasing ability, enabling the autobuilding of a protein structure that had previously failed to be built. Estimating the error in the merged reflection intensities requires the understanding and propagation of all of the sources of error arising from the measurements. One type of error, which is well understood, is the counting error introduced when the detector counts X-ray photons. Thus, if other types of random errors (such as readout noise) as well as uncertainties in systematic corrections (such as from X-ray attenuation) are completely understood, they can be propagated along with the counting error, as appropriate. In practice, most software packages propagate as much error as they know how to model and then include error-adjustment terms that scale the error estimates until they explain the variance among the measurements. If this is performed carefully, then during SAD phasing likelihood-based approaches can make optimal use of these error estimates, increasing the chance of a successful structure solution. In serial crystallography, SAD phasing has remained challenging, with the few examples of de novo protein structure solution each requiring many thousands of diffraction patterns. Here, the effects of different methods of treating the error estimates are estimated and it is shown that using a parametric approach that includes terms proportional to the known experimental uncertainty, the reflection intensity and the squared reflection intensity to improve the error estimates can allow SAD phasing even from weak zinc anomalous signal.
Assuntos
Cristalografia por Raios X/métodos , Modelos Moleculares , Termolisina/química , Cristalização/métodos , Interpretação Estatística de Dados , Conjuntos de Dados como Assunto , Funções VerossimilhançaRESUMO
Harnessing metal-free photoinduced reversible-deactivation radical polymerization (photo-RDRP) in organic and aqueous phases, we report a synthetic approach to enzyme-responsive and pro-apoptotic peptide brush polymers. Thermolysin-responsive peptide-based polymeric amphiphiles assembled into spherical micellar nanoparticles that undergo a morphology transition to worm-like micelles upon enzyme-triggered cleavage of coronal peptide sidechains. Moreover, pro-apoptotic polypeptide brushes show enhanced cell uptake over individual peptide chains of the same sequence, resulting in a significant increase in cytotoxicity to cancer cells. Critically, increased grafting density of pro-apoptotic peptides on brush polymers correlates with increased uptake efficiency and concurrently, cytotoxicity. The mild synthetic conditions afforded by photo-RDRP, make it possible to access well-defined peptide-based polymer bioconjugate structures with tunable bioactivity.
Assuntos
Micelas , Nanopartículas/química , Peptídeos/química , Polímeros/química , Termolisina/química , Acrilatos/química , Aminoácidos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Radicais Livres/química , Células HeLa , Humanos , Conformação Molecular , Processos Fotoquímicos , Polimerização , Polimetil Metacrilato/química , Solventes/química , Relação Estrutura-AtividadeRESUMO
The ability to precisely control the localization of enzymes on a surface is critical for several applications including biosensing, bionanoreactors, and single molecule studies. Despite recent advances, fabrication of enzyme patterns with resolution at the single enzyme level is limited by the lack of lithography methods that combine high resolution, compatibility with soft, polymeric structures, ease of fabrication, and high throughput. Here, a method to generate enzyme nanopatterns (using thermolysin as a model system) on a polymer surface is demonstrated using thermochemical scanning probe lithography (tc-SPL). Electrostatic immobilization of negatively charged sulfonated enzymes occurs selectively at positively charged amine nanopatterns produced by thermal deprotection of amines along the side-chain of a methacrylate-based copolymer film via tc-SPL. This process occurs simultaneously with local thermal quasi-3D topographical patterning of the same polymer, offering lateral sub-10 nm resolution, and vertical 1 nm resolution, as well as high throughput (5.2 × 104 µm2/h). The obtained single-enzyme resolution patterns are characterized by atomic force microscopy (AFM) and fluorescence microscopy. The enzyme density, the surface passivation, and the quasi-3D arbitrary geometry of these patterned pockets are directly controlled during the tc-SPL process in a single step without the need of markers or masks. Other unique features of this patterning approach include the combined single-enzyme resolution over mm2 areas and the possibility of fabricating enzymes nanogradients.
Assuntos
Nanotecnologia/métodos , Termolisina/química , Aminas/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Metacrilatos/química , Microscopia de Força Atômica , Nanoestruturas/química , Polímeros/química , Propriedades de Superfície , Termolisina/metabolismoRESUMO
High-throughput and large-scale patterning of enzymes with sub-10 nm resolution, the size range of individual protein molecules, is crucial for propelling advancement in a variety of areas, from the development of chip-based biomolecular nano-devices to molecular-level studies of cell biology. Despite recent developments in bio-nanofabrication technology, combining 10 nm resolution with high-throughput and large-scale patterning of enzymes is still an open challenge. Here, we demonstrate a high resolution and high-throughput patterning method to generate enzyme nanopatterns with sub-10 nm resolution by using thermochemical scanning probe lithography (tc-SPL). First, tc-SPL is used to generate amine patterns on a methacrylate copolymer film. Thermolysin enzymes functionalized with sulfonate-containing fluorescent labels (Alexa-488) are then directly immobilized onto the amine patterns through electrostatic interaction. Enzyme patterns with sub-10 nm line width are obtained as evidenced by atomic force microscopy (AFM) and fluorescence microscopy. Moreover, we demonstrate large-scale and high throughput (0.13 × 0.1 mm2 at a throughput of 5.2 × 104 µm2 h-1) patterning of enzymes incorporating 10 nm detailed pattern features. This straightforward and high-throughput method of fabricating enzyme nanopatterns will have a significant impact on future bio-nanotechnology applications and molecular-level biological studies. By scaling up using parallel probes, tc-SPL is promising for implementation to scale up the fabrication of nano-biodevices.
Assuntos
Bacillus/enzimologia , Bioimpressão/métodos , Enzimas Imobilizadas/química , Termolisina/química , Aminação , Bacillus/química , Corantes Fluorescentes/química , Metacrilatos/química , Nanotecnologia/métodos , Eletricidade EstáticaRESUMO
Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.
Assuntos
Cristalografia por Raios X/métodos , Coleta de Dados/métodos , Proteínas/química , Software , Animais , Cristalografia por Raios X/economia , Cristalografia por Raios X/instrumentação , Coleta de Dados/economia , Coleta de Dados/instrumentação , Humanos , Muramidase/química , Conformação Proteica , Receptor Muscarínico M2/química , Termolisina/químicaRESUMO
Single-wavelength anomalous diffraction (SAD) phasing from multiple crystals can be especially challenging in samples with weak anomalous signals and/or strong non-isomorphism. Here, advantage is taken of the combinatorial diversity possible in such experiments to study the relationship between merging statistics and downstream metrics of phasing signals. It is furthermore shown that a genetic algorithm (GA) can be used to optimize the grouping of data sets to enhance weak anomalous signals based on these merging statistics.
Assuntos
Cristalografia por Raios X/métodos , Coleta de Dados/métodos , Algoritmos , Bacillus/química , Proteínas de Bactérias/química , Cristalização/métodos , Modelos Moleculares , Conformação Proteica , Sporosarcina/química , Termolisina/química , Urease/químicaRESUMO
Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.
Assuntos
Nucleotídeos de Desoxiguanina/química , Pirofosfatases/química , Pirofosfatases/genética , Domínio Catalítico , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Temperatura , Termolisina/químicaRESUMO
The interaction of the amyloid-ß peptide (Aß) with thermolysin (TLN) was investigated by X-ray crystallography. Structural models of the complexes of TLN with several Aß fragments show that, despite the numerous possible cleavage sites of the Aß sequence, the C-terminal product of Ala30-Ile31 cleavage does not dissociate, thus inhibiting the enzyme. The high similarity between the TLN structural motif and neprilysin (NEP), the most extensively studied peptidase associated with Aß clearance, suggests that NEP should be more efficient against Aß polymorphs where Ala30-Ile31 is inaccessible, which is in agreement with studies in living mice that point to the limited role of NEP in degrading soluble Aß and its higher ability to degrade insoluble and/or oligomeric Aß forms, producing only the Aß10-37 intermediate.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Neprilisina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Termolisina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Neprilisina/química , Conformação Proteica , Proteólise , Termolisina/químicaRESUMO
Communicated by Ramaswamy H. Sarma.
Assuntos
Soluções/química , Termolisina/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Modelos Moleculares , Conformação ProteicaRESUMO
The Dispase autolysis-inducing protein (DAIP) is produced by Streptomyces mobaraensis to disarm neutral metalloproteases by decomposition. The absence of a catalytic protease domain led to the assumption that the seven-bladed ß-propeller protein DAIP causes structural modifications, thereby triggering autolysis. Determination of protein complexes consisting of DAIP and thermolysin or DAIP and a nonfunctional E138A bacillolysin variant supported this postulation. Protein twisting was indicated by DAIP-mediated inhibition of thermolysin while bacillolysin underwent immediate autolysis under the same conditions. Interestingly, an increase in SYPRO orange fluorescence allowed tracking of the fast degradation process. Similarly rapid autolysis of thermolysin mediated by DAIP was only observed upon the addition of amphiphilic compounds, which probably amplify the induced structural changes. DAIP further caused degradation of FITC-labeled E138A bacillolysin by trypsin, as monitored by a linear decrease in fluorescence polarization. The kinetic model, calculated from the obtained data, suggested a three-step mechanism defined by (a) fast DAIP-metalloprotease complex formation, (b) slower DAIP-mediated protein twisting, and (c) fragmentation. These results were substantiated by crystallized DAIP attached to a C-terminal helix fragment of thermolysin. Structural superposition of the complex with thermolysin is indicative of a conformational change upon binding to DAIP. Importantly, the majority of metalloproteases, also including homologs from various pathogens, are highly conserved at the autolysis-prone peptide bonds, suggesting their susceptibility to DAIP-mediated decomposition, which may offer opportunities for pharmaceutical applications. DATABASES: The atomic coordinates and structure factors (PDB ID: 6FHP) have been deposited in the Protein Data Bank (http://www.pdb.org/). ENZYMES: Aureolysin, EC 3.4.24.29; bacillolysin (Dispase, Gentlyase), EC 3.4.24.28; lasB (elastase), EC 3.4.24.4; subtilisin, EC 3.4.21.62; thermolysin, EC 3.4.24.27; transglutaminase, EC 2.3.2.13; trypsin, EC 3.4.21.4; vibriolysin (hemagglutinin(HA)/protease), EC 3.4.24.25.
Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Streptomyces/enzimologia , Termolisina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Catálise , Cristalografia por Raios X , Endopeptidases/química , Metaloendopeptidases/química , Metaloproteases/química , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Termolisina/químicaRESUMO
The DIALS diffraction-modeling software package has been applied to serial crystallography data. Diffraction modeling is an exercise in determining the experimental parameters, such as incident beam wavelength, crystal unit cell and orientation, and detector geometry, that are most consistent with the observed positions of Bragg spots. These parameters can be refined by nonlinear least-squares fitting. In previous work, it has been challenging to refine both the positions of the sensors (metrology) on multipanel imaging detectors such as the CSPAD and the orientations of all of the crystals studied. Since the optimal models for metrology and crystal orientation are interdependent, alternate cycles of panel refinement and crystal refinement have been required. To simplify the process, a sparse linear algebra technique for solving the normal equations was implemented, allowing the detector panels to be refined simultaneously against the diffraction from thousands of crystals with excellent computational performance. Separately, it is shown how to refine the metrology of a second CSPAD detector, positioned at a distance of 2.5â m from the crystal, used for recording low-angle reflections. With the ability to jointly refine the detector position against the ensemble of all crystals used for structure determination, it is shown that ensemble refinement greatly reduces the apparent nonisomorphism that is often observed in the unit-cell distributions from still-shot serial crystallography. In addition, it is shown that batching the images by timestamp and re-refining the detector position can realistically model small, time-dependent variations in detector position relative to the sample, and thereby improve the integrated structure-factor intensity signal and heavy-atom anomalous peak heights.