RESUMEN
Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings.
Asunto(s)
Péptidos , Olfato , Péptidos/química , Conformación Proteica en Hélice alfaRESUMEN
The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I-K-E-I-A-x-Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.
RESUMEN
The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.
Asunto(s)
Canales Iónicos/química , Membrana Dobles de Lípidos/química , Péptidos/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa , Ingeniería de Proteínas , Solubilidad , Agua/químicaRESUMEN
Rational protein design requires understanding the contribution of each amino acid to a targeted protein fold. For a subset of protein structures, namely, α-helical coiled coils (CCs), knowledge is sufficiently advanced to allow the rational de novo design of many structures, including entirely new protein folds. Current CC design rules center on using aliphatic hydrophobic residues predominantly to drive the folding and assembly of amphipathic α helices. The consequences of using aromatic residues-which would be useful for introducing structural probes, and binding and catalytic functionalities-into these interfaces are not understood. There are specific examples of designed CCs containing such aromatic residues, e.g., phenylalanine-rich sequences, and the use of polar aromatic residues to make buried hydrogen-bond networks. However, it is not known generally if sequences rich in tyrosine can form CCs, or what CC assemblies these would lead to. Here, we explore tyrosine-rich sequences in a general CC-forming background and resolve new CC structures. In one of these, an antiparallel tetramer, the tyrosine residues are solvent accessible and pack at the interface between the core and the surface. In another more complex structure, the residues are buried and form an extended hydrogen-bond network.
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Pliegue de Proteína , Proteínas , Secuencia de Aminoácidos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Secundaria de ProteínaRESUMEN
De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.
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Péptidos/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Conformación Proteica , Conformación Proteica en Hélice alfa , Ingeniería de Proteínas , Proteínas/química , SolventesRESUMEN
The association of amphipathic α helices in water leads to α-helical-bundle protein structures. However, the driving force for this-the hydrophobic effect-is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle-the α-helical coiled coils-relationships have been established that discriminate between all-parallel dimers, trimers, and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high resolution with X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of α-helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.
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Proteínas/química , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Agua/químicaRESUMEN
Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis are pathogenic bacteria adapted to reside on human respiratory mucosal epithelia. One common feature of these species is their ability to target members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, especially CEACAM1, which is achieved via structurally distinct ligands expressed by each species. Beside respiratory epithelial cells, cells at the dentogingival junction express high levels of CEACAM1. It is possible that bacterial species resident within the oral cavity also utilise CEACAM1 for colonisation and invasion of gingival tissues. From a screen of 59 isolates from the human oral cavity representing 49 bacterial species, we identified strains from Fusobacterium bound to CEACAM1. Of the Fusobacterium species tested, the CEACAM1-binding property was exhibited by F. nucleatum (Fn) and F. vincentii (Fv) but not F. polymorphum (Fp) or F. animalis (Fa) strains tested. These studies identified that CEACAM adhesion was mediated using a trimeric autotransporter adhesin (TAA) for which no function has thus far been defined. We therefore propose the name CEACAM binding protein of Fusobacterium (CbpF). CbpF was identified to be present in the majority of unspeciated Fusobacterium isolates confirming a subset of Fusobacterium spp. are able to target human CEACAM1.
RESUMEN
In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two-four helices and cyclic (Cn) or dihedral (Dn) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of ß-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs-namely, knobs-into-holes packing of side chains between helices-is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.
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Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Agua/químicaRESUMEN
The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT48-57) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.
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Genes jun , Péptidos/química , Proteínas Proto-Oncogénicas c-fos/química , Proto-Oncogenes , Secuencia de Aminoácidos , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dicroismo Circular , Femenino , Humanos , Modelos Moleculares , Péptidos/genética , Péptidos/farmacología , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/farmacología , TermodinámicaRESUMEN
MOTIVATION: The rational design of biomolecules is becoming a reality. However, further computational tools are needed to facilitate and accelerate this, and to make it accessible to more users. RESULTS: Here we introduce ISAMBARD, a tool for structural analysis, model building and rational design of biomolecules. ISAMBARD is open-source, modular, computationally scalable and intuitive to use. These features allow non-experts to explore biomolecular design in silico. ISAMBARD addresses a standing issue in protein design, namely, how to introduce backbone variability in a controlled manner. This is achieved through the generalization of tools for parametric modelling, describing the overall shape of proteins geometrically, and without input from experimentally determined structures. This will allow backbone conformations for entire folds and assemblies not observed in nature to be generated de novo, that is, to access the 'dark matter of protein-fold space'. We anticipate that ISAMBARD will find broad applications in biomolecular design, biotechnology and synthetic biology. AVAILABILITY AND IMPLEMENTATION: A current stable build can be downloaded from the python package index (https://pypi.python.org/pypi/isambard/) with development builds available on GitHub (https://github.com/woolfson-group/) along with documentation, tutorial material and all the scripts used to generate the data described in this paper. CONTACT: d.n.woolfson@bristol.ac.uk or chris.wood@bristol.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Conformación Proteica , Programas Informáticos , Simulación por Computador , Modelos Moleculares , Pliegue de Proteína , Proteínas/químicaRESUMEN
The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.
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Hidrolasas de Éster Carboxílico/química , Ingeniería de Proteínas , Hidrolasas de Éster Carboxílico/genética , Catálisis , Dominio Catalítico , Hidrólisis , Cinética , Mutación , Nitrofenoles/química , Conformación Proteica en Hélice alfa , Ingeniería de Proteínas/métodos , Estructura Terciaria de ProteínaRESUMEN
An ability to design peptide-based nanotubes (PNTs) rationally with defined and mutable internal channels would advance understanding of peptide self-assembly, and present new biomaterials for nanotechnology and medicine. PNTs have been made from Fmoc dipeptides, cyclic peptides, and lock-washer helical bundles. Here we show that blunt-ended α-helical barrels, that is, preassembled bundles of α-helices with central channels, can be used as building blocks for PNTs. This approach is general and systematic, and uses a set of de novo helical bundles as standards. One of these bundles, a hexameric α-helical barrel, assembles into highly ordered PNTs, for which we have determined a structure by combining cryo-transmission electron microscopy, X-ray fiber diffraction, and model building. The structure reveals that the overall symmetry of the peptide module plays a critical role in ripening and ordering of the supramolecular assembly. PNTs based on pentameric, hexameric, and heptameric α-helical barrels sequester hydrophobic dye within their lumens.
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Nanotecnología/métodos , Nanotubos de Péptidos/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Polimerizacion , Estructura Secundaria de Proteína , Desplegamiento Proteico , TemperaturaRESUMEN
The design of protein sequences that fold into prescribed de novo structures is challenging. General solutions to this problem require geometric descriptions of protein folds and methods to fit sequences to these. The α-helical coiled coils present a promising class of protein for this and offer considerable scope for exploring hitherto unseen structures. For α-helical barrels, which have more than four helices and accessible central channels, many of the possible structures remain unobserved. Here, we combine geometrical considerations, knowledge-based scoring, and atomistic modeling to facilitate the design of new channel-containing α-helical barrels. X-ray crystal structures of the resulting designs match predicted in silico models. Furthermore, the observed channels are chemically defined and have diameters related to oligomer state, which present routes to design protein function.
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Biología Computacional , Modelos Moleculares , Estructura Secundaria de Proteína , Agua/química , Péptidos/química , SolubilidadRESUMEN
MOTIVATION: The ability to accurately model protein structures at the atomistic level underpins efforts to understand protein folding, to engineer natural proteins predictably and to design proteins de novo. Homology-based methods are well established and produce impressive results. However, these are limited to structures presented by and resolved for natural proteins. Addressing this problem more widely and deriving truly ab initio models requires mathematical descriptions for protein folds; the means to decorate these with natural, engineered or de novo sequences; and methods to score the resulting models. RESULTS: We present CCBuilder, a web-based application that tackles the problem for a defined but large class of protein structure, the α-helical coiled coils. CCBuilder generates coiled-coil backbones, builds side chains onto these frameworks and provides a range of metrics to measure the quality of the models. Its straightforward graphical user interface provides broad functionality that allows users to build and assess models, in which helix geometry, coiled-coil architecture and topology and protein sequence can be varied rapidly. We demonstrate the utility of CCBuilder by assembling models for 653 coiled-coil structures from the PDB, which cover >96% of the known coiled-coil types, and by generating models for rarer and de novo coiled-coil structures. AVAILABILITY AND IMPLEMENTATION: CCBuilder is freely available, without registration, at http://coiledcoils.chm.bris.ac.uk/app/cc_builder/.
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Modelos Moleculares , Estructura Secundaria de Proteína , Programas Informáticos , Secuencia de Aminoácidos , Internet , Ingeniería de Proteínas , Pliegue de ProteínaRESUMEN
Ab initio design of enzymes requires precise and predictable positioning of reactive functional groups within accessible and controlled environments of de novo protein scaffolds. Here we show that multiple thiol moieties can be placed within a central channel, with approximate dimensions 6 × 42 Å, of a de novo, six-helix peptide assembly (CC-Hex). Layers of six cysteine residues are introduced at two different sites ~6 (the "L24C" mutant) and ~17 Å (L17C) from the C-terminal opening of the channel. X-ray crystal structures confirm the mutant structures as hexamers with internal free thiol, rather than disulfide-linked cysteine residues. Both mutants are hexa-alkylated upon addition of iodoacetamide, demonstrating accessibility and full reactivity of the thiol groups. Comparison of the alkylation and unfolding rates of the hexamers indicates that access is directly through the channel and not via dissociation and unfolding of the assembly. Moreover, neither mutant reacts with iodoacetic acid, demonstrating selectivity of the largely hydrophobic channel. These studies show that it is possible to engineer reactive side chains with both precision and control into a de novo scaffold to produce protein-like structures with chemoselective reactivity.
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Péptidos/síntesis química , Teoría Cuántica , Cristalografía por Rayos X , Cisteína/química , Modelos Moleculares , Péptidos/química , Desplegamiento Proteico , Compuestos de Sulfhidrilo/químicaRESUMEN
Nature presents various protein fibers that bridge the nanometer to micrometer regimes. These structures provide inspiration for the de novo design of biomimetic assemblies, both to address difficulties in studying and understanding natural systems, and to provide routes to new biomaterials with potential applications in nanotechnology and medicine. We have designed a self-assembling fiber system, the SAFs, in which two small α-helical peptides are programmed to form a dimeric coiled coil and assemble in a controlled manner. The resulting fibers are tens of nm wide and tens of µm long, and, therefore, comprise millions of peptides to give gigadalton supramolecular structures. Here, we describe the structure of the SAFs determined to approximately 8 Å resolution using cryotransmission electron microscopy. Individual micrographs show clear ultrastructure that allowed direct interpretation of the packing of individual α-helices within the fibers, and the construction of a 3D electron density map. Furthermore, a model was derived using the cryotransmission electron microscopy data and side chains taken from a 2.3 Å X-ray crystal structure of a peptide building block incapable of forming fibers. This was validated using single-particle analysis techniques, and was stable in prolonged molecular-dynamics simulation, confirming its structural viability. The level of self-assembly and self-organization in the SAFs is unprecedented for a designed peptide-based material, particularly for a system of considerably reduced complexity compared with natural proteins. This structural insight is a unique high-resolution description of how α-helical fibrils pack into larger protein fibers, and provides a basis for the design and engineering of future biomaterials.
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Microscopía por Crioelectrón/métodos , Péptidos/química , Cristalografía por Rayos X , Secciones por Congelación , Modelos Moleculares , Peso Molecular , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.
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Péptidos/química , Secuencia de Aminoácidos , Fenómenos Biofísicos , Diseño Asistido por Computadora , Cristalografía por Rayos X , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Biología SintéticaRESUMEN
CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.
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Antifúngicos/metabolismo , Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Econazol/metabolismo , Lepra/tratamiento farmacológico , Mycobacterium leprae , Antifúngicos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Econazol/farmacología , Hemo/metabolismo , Histidina/metabolismo , Humanos , Hierro/metabolismo , Cinética , Lepra/microbiología , Modelos Moleculares , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/enzimología , Mycobacterium smegmatis/química , Mycobacterium smegmatis/enzimología , Porfirinas/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6-Å channel is permeable to water. One layer of leucine residues within the channel is mutable, accepting polar aspartic acid and histidine side chains, which leads to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)(3) heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.
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Oligopéptidos/química , Biología Sintética , Ácido Aspártico/química , Cristalografía por Rayos X , Histidina/química , Modelos Moleculares , Oligopéptidos/síntesis química , Conformación Proteica , Ingeniería de ProteínasRESUMEN
Bacterial cell surfaces are commonly decorated with a layer formed from multiple copies of adhesin proteins whose binding interactions initiate colonization and infection processes. In this study, we investigate the physical deformability of the UspA1 adhesin protein from Moraxella catarrhalis, a causative agent of middle-ear infections in humans. UspA1 binds a range of extracellular proteins including fibronectin, and the epithelial cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Electron microscopy indicates that unliganded UspA1 is densely packed at, and extends about 800 Å from, the Moraxella surface. Using a modified atomic force microscope, we show that the adhesive properties and thickness of the UspA1 layer at the cell surface varies on addition of either fibronectin or CEACAM1. This in situ analysis is then correlated with the molecular structure of UspA1. To provide an overall model for UspA1, we have determined crystal structures for two N-terminal fragments which are then combined with a previous structure of the CEACAM1-binding site. We show that the UspA1-fibronectin complex is formed between UspA1 head region and the 13th type-III domain of fibronectin and, using X-ray scattering, that the complex involves an angular association between these two proteins. In combination with a previous study, which showed that the CEACAM1-UspA1 complex is distinctively bent in solution, we correlate these observations on isolated fragments of UspA1 with its in situ response on the cell surface. This study therefore provides a rare direct demonstration of protein conformational change at the cell surface.