RESUMEN
Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13-amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.
Asunto(s)
Hidrogenasas , Níquel , Níquel/química , Níquel/metabolismo , Hidrogenasas/química , Protones , Hidrógeno/química , PéptidosRESUMEN
The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.
RESUMEN
In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo. While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S]2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Ingeniería de Proteínas/métodos , Secuencias de Aminoácidos , Dicroismo Circular , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Mutagénesis , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Conformación Proteica en Hélice alfaRESUMEN
Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO2, is competitively displaced by O2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO2 for O2 Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO2 and O2 Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO2 This paper identifies a key structural feature that enabled the enzyme to evolve improved CO2 sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.
Asunto(s)
Fenómenos Biofísicos , Modelos Moleculares , Conformación Proteica , Ribulosa-Bifosfato Carboxilasa/química , Dióxido de Carbono/química , Catálisis , Modelos Químicos , Simulación de Dinámica Molecular , Filogenia , Ribulosa-Bifosfato Carboxilasa/clasificación , Ribulosa-Bifosfato Carboxilasa/genética , Análisis Espectral , Especificidad por SustratoRESUMEN
We explore the capacity of the de novo protein, S824, to incorporate a multinuclear iron-sulfur cluster within the core of a single-chain four-helix bundle. This topology has a high intrinsic designability because sequences are constrained largely by the pattern of hydrophobic and hydrophilic amino acids, thereby allowing for the extensive substitution of individual side chains. Libraries of novel proteins based on these constraints have surprising functional potential and have been shown to complement the deletion of essential genes in E. coli. Our structure-based design of four first-shell cysteine ligands, one per helix, in S824 resulted in successful incorporation of a cubane Fe4 S4 cluster into the protein core. A number of challenges were encountered during the design and characterization process, including nonspecific metal-induced aggregation and the presence of competing metal-cluster stoichiometries. The introduction of buried iron-sulfur clusters into the helical bundle is an initial step toward converting libraries of designed structures into functional de novo proteins with catalytic or electron-transfer functionalities.
Asunto(s)
Escherichia coli , Proteínas Hierro-Azufre , Ingeniería de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Conformación Proteica en Hélice alfaRESUMEN
Life on Earth is driven by electron transfer reactions catalyzed by a suite of enzymes that comprise the superfamily of oxidoreductases (Enzyme Classification EC1). Most modern oxidoreductases are complex in their structure and chemistry and must have evolved from a small set of ancient folds. Ancient oxidoreductases from the Archean Eon between ca. 3.5 and 2.5 billion years ago have been long extinct, making it challenging to retrace evolution by sequence-based phylogeny or ancestral sequence reconstruction. However, three-dimensional topologies of proteins change more slowly than sequences. Using comparative structure and sequence profile-profile alignments, we quantify the similarity between proximal cofactor-binding folds and show that they are derived from a common ancestor. We discovered that two recurring folds were central to the origin of metabolism: ferredoxin and Rossmann-like folds. In turn, these two folds likely shared a common ancestor that, through duplication, recruitment, and diversification, evolved to facilitate electron transfer and catalysis at a very early stage in the origin of metabolism.
Asunto(s)
Transporte de Electrón , Evolución Molecular , Oxidorreductasas/metabolismo , Ferredoxinas/metabolismo , Flavodoxina/metabolismo , Conformación ProteicaRESUMEN
Ambidoxin is a designed, minimal dodecapeptide consisting of alternating L and D amino acids that binds a 4Fe-4S cluster through ligand-metal interactions and an extensive network of second-shell hydrogen bonds. The peptide can withstand hundreds of oxidation-reduction cycles at room temperature. Ambidoxin suggests how simple, prebiotic peptides may have achieved robust redox catalysis on the early Earth.
Asunto(s)
Proteínas Hierro-Azufre/química , Técnicas Electroquímicas , Transporte de Electrón , Ligandos , Oxidación-ReducciónRESUMEN
To what extent do structural and biophysical features of food allergen proteins distinguish them from other proteins in our diet? Invertebrate tropomyosins (Tpms) as a class are considered "pan-allergens," inducing food allergy to shellfish and respiratory allergy to dust mites. Vertebrate Tpms are not known to elicit allergy or cross-reactivity, despite their high structural similarity and sequence identity to invertebrate homologs. We expect allergens are sufficiently stable against gastrointestinal proteases to survive for immune sensitization in the intestines, and that proteolytic stability will correlate with thermodynamic stability. Thermal denaturation of shrimp Tpm shows that it is more stable than non-allergen vertebrate Tpm. Shrimp Tpm is also more resistant to digestion. Molecular dynamics uncover local dynamics that select epitopes and global differences in flexibility between shrimp and pig Tpm that discriminate allergens from non-allergens. Molecular determinants of allergenicity depend not only on sequence but on contributions of protein structure and dynamics.
Asunto(s)
Alérgenos/química , Penaeidae/metabolismo , Porcinos/metabolismo , Tropomiosina/química , Alérgenos/inmunología , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/inmunología , Reacciones Cruzadas , Epítopos/inmunología , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Penaeidae/inmunología , Desnaturalización Proteica , Homología de Secuencia de Aminoácido , Hipersensibilidad a los Mariscos/metabolismo , Tropomiosina/inmunologíaRESUMEN
Oxidoreductases catalyze electron transfer reactions that ultimately provide the energy for life. A limited set of ancestral protein-metal modules are presumably the building blocks that evolved into this diverse protein family. However, the identity of these modules and their path to modern oxidoreductases is unknown. Using a comparative structural analysis approach, we identify a set of fundamental electron transfer modules that have evolved to form the extant oxidoreductases. Using transition metal-containing cofactors as fiducial markers, it is possible to cluster cofactor microenvironments into as few as four major modules: bacterial ferredoxin, cytochrome c, symerythrin, and plastocyanin-type folds. From structural alignments, it is challenging to ascertain whether modules evolved from a single common ancestor (homology) or arose by independent convergence on a limited set of structural forms (analogy). Additional insight into common origins is contained in the spatial adjacency network (SPAN), which is based on proximity of modules in oxidoreductases containing multiple cofactor electron transfer chains. Electron transfer chains within complex modern oxidoreductases likely evolved through repeated duplication and diversification of ancient modular units that arose in the Archean eon.
Asunto(s)
Coenzimas/metabolismo , Evolución Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Coenzimas/química , Citocromos c/química , Citocromos c/metabolismo , Transporte de Electrón , Ferredoxinas/química , Ferredoxinas/metabolismo , Metales/química , Metales/metabolismo , Modelos Moleculares , Plastocianina/química , Plastocianina/metabolismo , Conformación Proteica , Homología Estructural de ProteínaRESUMEN
The ability to tune the light-absorption properties of chlorophylls by their protein environment is the key to the robustness and high efficiency of photosynthetic light-harvesting proteins. Unfortunately, the intricacy of the natural complexes makes it very difficult to identify and isolate specific protein-pigment interactions that underlie the spectral-tuning mechanisms. Herein we identify and demonstrate the tuning mechanism of chlorophyll spectra in typeâ II water-soluble chlorophyll binding proteins from Brassicaceae (WSCPs). By comparing the molecular structures of two natural WSCPs we correlate a shift in the chlorophyll red absorption band with deformation of its tetrapyrrole macrocycle that is induced by changing the position of a nearby tryptophan residue. We show by a set of reciprocal point mutations that this change accounts for up to 2/3 of the observed spectral shift between the two natural variants.
Asunto(s)
Proteínas de Unión a Clorofila/química , Clorofila/química , Luz , Brassicaceae/química , Proteínas de Unión a Clorofila/genética , Modelos Moleculares , Estructura Molecular , Mutación PuntualRESUMEN
Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aß. When the fused CP4-Aß construct assembled into antiparallel ß-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aß assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.
Asunto(s)
Péptidos/síntesis química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/química , Catálisis , Dominio Catalítico , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Péptidos/química , Multimerización de Proteína , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/químicaRESUMEN
We investigate formation of nano- to microscale peptide fibers and sheets where assembly requires association of two distinct collagen mimetic peptides (CMPs). The multicomponent nature of these designs allows the decoupling of amino acid contributions to peptide folding versus higher-order assembly. While both arginine and lysine containing CMP sequences can favor triple-helix folding, only arginine promotes rapid supramolecular assembly in each of the three two-component systems examined. Unlike lysine, the polyvalent guanidyl group of arginine is capable of both intra- and intermolecular contacts, promoting assembly. This is consistent with the supramolecular diversity of CMP morphologies observed throughout the literature. It also connects CMP self-assembly with a broad range of biomolecular interaction phenomena, providing general principles for modeling and design.
Asunto(s)
Colágeno/química , Lisina/química , Modelos Moleculares , Péptidos/química , Secuencia de Aminoácidos , Biomimética , Electricidad EstáticaRESUMEN
Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases. Controlling chemical transformations mediated by iron-sulfur centers such as nitrogen fixation, hydrogen production as well as electron transfer reactions involved in photosynthesis are of tremendous importance for sustainable chemistry and energy production initiatives. As such, there is significant interest in the design of iron-sulfur proteins as minimal models to gain fundamental understanding of complex natural systems and as lead-molecules for industrial and energy applications. Herein, we discuss salient structural characteristics of natural iron-sulfur proteins and how they guide principles for design. Model structures of past designs are analyzed in the context of these principles and potential directions for enhanced designs are presented, and new areas of iron-sulfur protein design are proposed. This article is part of a Special issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, protein networks, edited by Ronald L. Koder and J.L Ross Anderson.
Asunto(s)
Dominio Catalítico , Proteínas Hierro-Azufre/química , Metaloproteínas/química , Ingeniería de Proteínas/métodos , Dominio Catalítico/genética , Biología Computacional , Ferredoxinas/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Hierro/química , Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Azufre/química , Azufre/metabolismoRESUMEN
We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.
Asunto(s)
Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Metales/química , Metales/metabolismo , Imitación Molecular , Secuencia de Aminoácidos , Animales , Bovinos , Cobre/química , Cobre/metabolismo , Cobre/farmacología , Metaloproteínas/química , Metaloproteínas/genética , Metales/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/genética , Ingeniería de Proteínas , Estabilidad Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
One of the key challenges in modeling protein energetics is the treatment of solvent interactions. This is particularly important in the case of peptides, where much of the molecule is highly exposed to solvent due to its small size. In this study, we develop an empirical method for estimating the local dielectric constant based on an additive model of atomic polarizabilities. Calculated values match reported apparent dielectric constants for a series of Staphylococcus aureus nuclease mutants. Calculated constants are used to determine screening effects on Coulombic interactions and to determine solvation contributions based on a modified Generalized Born model. These terms are incorporated into the protein modeling platform protCAD, and benchmarked on a data set of collagen mimetic peptides for which experimentally determined stabilities are available. Computing local dielectric constants using atomistic protein models and the assumption of additive atomic polarizabilities is a rapid and potentially useful method for improving electrostatics and solvation calculations that can be applied in the computational design of peptides.
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Materiales Biomiméticos/química , Colágeno/química , Modelos Químicos , Modelos Moleculares , Péptidos/químicaRESUMEN
Type-I collagen self-assembles into a fibrillar gel at physiological temperature and pH to provide a cell-adhesive, supportive, structural network. As such, it is an attractive, popular scaffold for in vitro evaluations of cellular behavior and for tissue engineering applications. In this study, type-I collagen is modified to introduce methacrylate groups on the free amines of the lysine residues to create collagen methacrylamide (CMA). CMA retains the properties of collagen such as self-assembly, biodegradability, and natural bioactivity but is also photoactive and can be rapidly cross-linked or functionalized with acrylated molecules when irradiated with ultraviolet light in the presence of a photoinitiator. CMA also demonstrates unique temperature-dependent behavior. For natural type-I collagen, the overall structure of the fiber network remains largely static over time scales of a few hours upon heating and cooling at temperatures below its denaturation point. CMA, however, is rapidly thermoreversible and will oscillate between a liquid macromer suspension and a semisolid fibrillar hydrogel when the temperature is modulated between 10 and 37 °C. Using a series of mechanical, scattering, and spectroscopic methods, we demonstrate that structural reversibility is manifest across multiple scales from the protein topology of the triple helix up through the rheological properties of the CMA hydrogel. Electron microscopy imaging of CMA after various stages of heating and cooling shows that the canonical collagen-like D-periodic banding ultrastructure of the fibers is preserved. A rapidly thermoreversible collagen-based hydrogel is expected to have wide utility in tissue engineering and drug delivery applications as a biofunctional, biocompatible material. Thermal reversibility also makes CMA a powerful model for studying the complex process of hierarchical collagen self-assembly.
Asunto(s)
Acrilamidas/síntesis química , Colágeno Tipo I/síntesis química , Metacrilatos/química , Temperatura , Acrilamidas/química , Colágeno Tipo I/química , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.
Asunto(s)
Ritmo Circadiano/fisiología , Drosophila/fisiología , Actividad Motora/fisiología , Sueño/fisiología , AnimalesRESUMEN
BACKGROUND: Immune challenge impacts behavior in many species. In mammals, this adaptive behavior is often manifested as an increase in sleep. Sleep has therefore been proposed to benefit the host by enhancing immune function and thereby overcome the challenge. To facilitate genetic studies on the relationship between sleep and immune function, we characterized the effect of the immune response on sleep in Drosophila melanogaster. Behavioral features of sleep as well as the innate immune response signaling pathways are well characterized in flies and are highly conserved in mammals. RESULTS: An immune response induced by infection with Gram-negative bacteria or by aseptic injury increased sleep in flies. The increase in sleep occurred during the morning hours after treatment and the magnitude of the effect was dependent on the time-of-day of inoculation or injury such that night-time treatment had a stronger effect than that during the daytime. This pattern persisted in constant darkness, indicating a role of the circadian clock. Mutants of the circadian clock gene, period, eliminated the increase in sleep observed in the morning, but instead showed enhanced sleep immediately after injury or infection.Null mutants of the Nuclear Factor kappaB (NFkappaB) Relish, which is central to the innate immune response, do not increase sleep in response to injury or infection at any time of day. Instead, they maintain a normal sleep pattern until they die. Expression of a full-length Relish transgene in the fat bodies of Relish mutants restored the morning increase in sleep during an immune response. Fat bodies are a major site of immune signalling in flies and have a key role in host defense. CONCLUSIONS: These data demonstrate that an immune response increases sleep in flies in a manner that is gated by the circadian clock and that requires the NFkappaB Relish. These findings support a role of sleep in a recovery process and demonstrate a conserved feature of the Drosophila model of sleep.