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1.
Nucleic Acids Res ; 50(14): 7829-7841, 2022 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-35880577

RESUMEN

The kinetics of DNA hybridization are fundamental to biological processes and DNA-based technologies. However, the precise physical mechanisms that determine why different DNA sequences hybridize at different rates are not well understood. Secondary structure is one predictable factor that influences hybridization rates but is not sufficient on its own to fully explain the observed sequence-dependent variance. In this context, we measured hybridization rates of 43 different DNA sequences that are not predicted to form secondary structure and present a parsimonious physically justified model to quantify our observations. Accounting only for the combinatorics of complementary nucleating interactions and their sequence-dependent stability, the model achieves good correlation with experiment with only two free parameters. Our results indicate that greater repetition of Watson-Crick pairs increases the number of initial states able to proceed to full hybridization, with the stability of those pairings dictating the likelihood of such progression, thus providing new insight into the physical factors underpinning DNA hybridization rates.


Asunto(s)
ADN , Conformación de Ácido Nucleico , ADN/química , Cinética , Hibridación de Ácido Nucleico , Termodinámica
2.
Angew Chem Int Ed Engl ; 62(51): e202314458, 2023 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-37903739

RESUMEN

Hierarchical DNA nanostructures offer programmable functions at scale, but making these structures dynamic, while keeping individual components intact, is challenging. Here we show that the DNA A-motif-protonated, self-complementary poly(adenine) sequences-can propagate DNA origami into one-dimensional, micron-length fibrils. When coupled to a small molecule pH regulator, visible light can activate the hierarchical assembly of our DNA origami into dissipative fibrils. This system is recyclable and does not require DNA modification. By employing a modular and waste-free strategy to assemble and disassemble hierarchical structures built from DNA origami, we offer a facile and accessible route to developing well-defined, dynamic, and large DNA assemblies with temporal control. As a general tool, we envision that coupling the A-motif to cycles of dissipative protonation will allow the transient construction of diverse DNA nanostructures, finding broad applications in dynamic and non-equilibrium nanotechnology.


Asunto(s)
Nanoestructuras , Conformación de Ácido Nucleico , Nanoestructuras/química , ADN/química , Nanotecnología/métodos , Citoesqueleto
3.
Nucleic Acids Res ; 47(22): 11963-11975, 2019 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-31728524

RESUMEN

DNA origami allows for the synthesis of nanoscale structures and machines with nanometre precision and high yields. Tubular DNA origami nanostructures are particularly useful because their geometry facilitates a variety of applications including nanoparticle encapsulation, the construction of artificial membrane pores and as structural scaffolds that can uniquely spatially arrange nanoparticles in circular, linear and helical arrays. Here we report a system of parametrization for the design of radially symmetric DNA origami nanotubes with adjustable diameter, length, crossover density, pleat angle and chirality. The system is implemented into a computational algorithm that provides a practical means to navigate the complex geometry of DNA origami nanotube design. We apply this in the design, synthesis and characterization of novel DNA origami nanotubes. These include structures with pleated walls where the same number of duplexes can form nanotubes with different diameters, and to vary the diameter within the same structure. We also construct nanotubes that can be reconfigured into different chiral shapes. Finally, we explore the effect of strain on the local and global geometry of DNA origami nanotubes and demonstrate how pleated walls can provide a strategy to rigidify nanotubes and to construct closely packed parallel duplexes.


Asunto(s)
ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Nanotubos/química , Conformación de Ácido Nucleico , Algoritmos , Secuencia de Bases , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Tamaño de la Partícula
4.
Nucleic Acids Res ; 44(3): 1411-20, 2016 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-26681693

RESUMEN

Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR-DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR-DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites.


Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cromatografía en Gel , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Difracción de Rayos X
5.
Nature ; 466(7309): 996-1000, 2010 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-20676082

RESUMEN

The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flagelos/química , Flagelos/fisiología , Proteínas Motoras Moleculares/química , Rotación , Torque , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Flagelos/genética , Modelos Moleculares , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , Relación Estructura-Actividad , Thermotoga maritima/química
6.
Proc Natl Acad Sci U S A ; 109(15): 5663-8, 2012 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-22451932

RESUMEN

The HIV-1 envelope (Env) spike (gp120(3)/gp41(3)) undergoes considerable structural rearrangements to mediate virus entry into cells and to evade the host immune response. Engagement of CD4, the primary human receptor, fixes a particular conformation and primes Env for entry. The CD4-bound state, however, is prone to spontaneous inactivation and susceptible to antibody neutralization. How does unliganded HIV-1 maintain CD4-binding capacity and regulate transitions to the CD4-bound state? To define this mechanistically, we determined crystal structures of unliganded core gp120 from HIV-1 clades B, C, and E. Notably, all of these unliganded HIV-1 structures resembled the CD4-bound state. Conformational fixation with ligand selection and thermodynamic analysis of full-length and core gp120 interactions revealed that the tendency of HIV-1 gp120 to adopt the CD4-bound conformation was restrained by the V1/V2- and V3-variable loops. In parallel, we determined the structure of core gp120 in complex with the small molecule, NBD-556, which specifically recognizes the CD4-bound conformation of gp120. Neutralization by NBD-556 indicated that Env spikes on primary isolates rarely assume the CD4-bound conformation spontaneously, although they could do so when quaternary restraints were loosened. Together, the results suggest that the CD4-bound conformation represents a "ground state" for the gp120 core, with variable loop and quaternary interactions restraining unliganded gp120 from "snapping" into this conformation. A mechanism of control involving deformations in unliganded structure from a functionally critical state (e.g., the CD4-bound state) provides advantages in terms of HIV-1 Env structural diversity and resistance to antibodies and inhibitors, while maintaining elements essential for entry.


Asunto(s)
Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Relación Estructura-Actividad
7.
J Biol Chem ; 288(21): 15269-79, 2013 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-23482564

RESUMEN

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.


Asunto(s)
Ácido Araquidónico/metabolismo , Artritis Reumatoide/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/metabolismo , Vimentina/metabolismo , Animales , Ácido Araquidónico/genética , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Células CHO , Cricetinae , Cricetulus , Diseño de Fármacos , Inhibidores Enzimáticos/uso terapéutico , Femenino , Fosfolipasas A2 Grupo II/genética , Fosfolipasas A2 Grupo II/metabolismo , Humanos , Masculino , Transducción de Señal/genética , Membrana Sinovial/patología , Vimentina/genética
8.
BMC Bioinformatics ; 14: 111, 2013 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-23530628

RESUMEN

BACKGROUND: Helical membrane proteins are vital for the interaction of cells with their environment. Predicting the location of membrane helices in protein amino acid sequences provides substantial understanding of their structure and function and identifies membrane proteins in sequenced genomes. Currently there is no comprehensive benchmark tool for evaluating prediction methods, and there is no publication comparing all available prediction tools. Current benchmark literature is outdated, as recently determined membrane protein structures are not included. Current literature is also limited to global assessments, as specialised benchmarks for predicting specific classes of membrane proteins were not previously carried out. DESCRIPTION: We present a benchmark server at http://sydney.edu.au/pharmacy/sbio/software/TMH_benchmark.shtml that uses recent high resolution protein structural data to provide a comprehensive assessment of the accuracy of existing membrane helix prediction methods. The server further allows a user to compare uploaded predictions generated by novel methods, permitting the comparison of these novel methods against all existing methods compared by the server. Benchmark metrics include sensitivity and specificity of predictions for membrane helix location and orientation, and many others. The server allows for customised evaluations such as assessing prediction method performances for specific helical membrane protein subtypes.We report results for custom benchmarks which illustrate how the server may be used for specialised benchmarks. Which prediction method is the best performing method depends on which measure is being benchmarked. The OCTOPUS membrane helix prediction method is consistently one of the highest performing methods across all measures in the benchmarks that we performed. CONCLUSIONS: The benchmark server allows general and specialised assessment of existing and novel membrane helix prediction methods. Users can employ this benchmark server to determine the most suitable method for the type of prediction the user needs to perform, be it general whole-genome annotation or the prediction of specific types of helical membrane protein. Creators of novel prediction methods can use this benchmark server to evaluate the performance of their new methods. The benchmark server will be a valuable tool for researchers seeking to extract more sophisticated information from the large and growing protein sequence databases.


Asunto(s)
Proteínas de la Membrana/química , Programas Informáticos , Benchmarking , Bases de Datos de Proteínas , Estructura Secundaria de Proteína , Análisis de Secuencia de Proteína
9.
ACS Nano ; 16(4): 6455-6467, 2022 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-35316035

RESUMEN

Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multivalent interactions to form stable complexes that are also capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multisite competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo, an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to benefit future studies that use DNA origami structures to exploit multivalent interactions for the design and synthesis of a wide range of possible kinetic behaviors.


Asunto(s)
Nanoestructuras , Nanotecnología , ADN/química , ADN de Cadena Simple , Nanoestructuras/química , Conformación de Ácido Nucleico
10.
ACS Nano ; 14(12): 17428-17441, 2020 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-33232603

RESUMEN

Biology demonstrates how a near infinite array of complex systems and structures at many scales can originate from the self-assembly of component parts on the nanoscale. But to fully exploit the benefits of self-assembly for nanotechnology, a crucial challenge remains: How do we rationally encode well-defined global architectures in subunits that are much smaller than their assemblies? Strain accumulation via geometric frustration is one mechanism that has been used to explain the self-assembly of global architectures in diverse and complex systems a posteriori. Here we take the next step and use strain accumulation as a rational design principle to control the length distributions of self-assembling polymers. We use the DNA origami method to design and synthesize a molecular subunit known as the PolyBrick, which perturbs its shape in response to local interactions via flexible allosteric blocking domains. These perturbations accumulate at the ends of polymers during growth, until the deformation becomes incompatible with further extension. We demonstrate that the key thermodynamic factors for controlling length distributions are the intersubunit binding free energy and the fundamental strain free energy, both which can be rationally encoded in a PolyBrick subunit. While passive polymerization yields geometrical distributions, which have the highest statistical length uncertainty for a given mean, the PolyBrick yields polymers that approach Gaussian length distributions whose variance is entirely determined by the strain free energy. We also show how strain accumulation can in principle yield length distributions that become tighter with increasing subunit affinity and approach distributions with uniform polymer lengths. Finally, coarse-grained molecular dynamics and Monte Carlo simulations delineate and quantify the dominant forces influencing strain accumulation in a molecular system. This study constitutes a fundamental investigation of the use of strain accumulation as a rational design principle in molecular self-assembly.

11.
Nanoscale ; 11(26): 12460-12464, 2019 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-31120079

RESUMEN

Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.


Asunto(s)
ADN de Cadena Simple/química , Nanotubos/química , Nanotubos/ultraestructura
12.
ACS Nano ; 12(6): 5791-5799, 2018 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-29812934

RESUMEN

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.


Asunto(s)
ADN/análisis , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Silicatos de Aluminio/química , Oro/química , Nanopartículas del Metal/química , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Propiedades de Superficie
13.
Methods Mol Biol ; 1593: 105-117, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28389948

RESUMEN

Small angle X-ray scattering is an increasingly utilized method for characterizing the shape and structural properties of proteins in solution. The technique is amenable to very large protein complexes and to dynamic particles with different conformational states. It is therefore ideally suited to the analysis of some flagellar motor components. Indeed, we recently used the method to analyze the solution structure of the flagellar motor protein FliG, which when combined with high-resolution snapshots of conformational states from crystal structures, led to insights into conformational transitions that are important in mediating the self-assembly of the bacterial flagellar motor. Here, we describe procedures for X-ray scattering data collection of flagellar motor components, data analysis, and interpretation.


Asunto(s)
Proteínas Bacterianas/química , Flagelos/química , Cristalografía por Rayos X/métodos , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Rayos X
14.
Sci Rep ; 7: 45180, 2017 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-28345591

RESUMEN

The droplet on hydrogel bilayer (DHB) is a novel platform for investigating the function of ion channels. Advantages of this setup include tight control of all bilayer components, which is compelling for the investigation of mechanosensitive (MS) ion channels, since they are highly sensitive to their lipid environment. However, the activation of MS ion channels in planar supported lipid bilayers, such as the DHB, has not yet been established. Here we present the activation of the large conductance MS channel of E. coli, (MscL), in DHBs. By selectively stretching the droplet monolayer with nanolitre injections of buffer, we induced quantifiable DHB tension, which could be related to channel activity. The MscL activity response revealed that the droplet monolayer tension equilibrated over time, likely by insertion of lipid from solution. Our study thus establishes a method to controllably activate MS channels in DHBs and thereby advances studies of MS channels in this novel platform.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Hidrogeles , Canales Iónicos/genética , Membrana Dobles de Lípidos/metabolismo , Gotas Lipídicas/metabolismo , Mutación
15.
Nat Struct Mol Biol ; 23(3): 197-203, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26854663

RESUMEN

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.


Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/química , Flagelos/química , Sustancias Macromoleculares/metabolismo , Proteínas Motoras Moleculares/metabolismo , Biogénesis de Organelos , Multimerización de Proteína , Proteínas Bacterianas/química , Sustancias Macromoleculares/química , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Conformación Proteica
16.
Nat Struct Mol Biol ; 22(7): 522-31, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26098315

RESUMEN

As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Antígenos CD4/inmunología , Cristalografía por Rayos X , Epítopos/inmunología , Células HEK293 , Infecciones por VIH/virología , VIH-1/química , VIH-1/inmunología , Humanos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Internalización del Virus
17.
J Biomol Struct Dyn ; 32(2): 308-18, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-23527746

RESUMEN

Major advances have been made in the prediction of soluble protein structures, led by the knowledge-based modeling methods that extract useful structural trends from known protein structures and incorporate them into scoring functions. The same cannot be reported for the class of transmembrane proteins, primarily due to the lack of high-resolution structural data for transmembrane proteins, which render many of the knowledge-based method unreliable or invalid. We have developed a method that harnesses the vast structural knowledge available in soluble protein data for use in the modeling of transmembrane proteins. At the core of the method, a set of transmembrane protein decoy sets that allow us to filter and train features recognized from soluble proteins for transmembrane protein modeling into a set of scoring functions. We have demonstrated that structures of soluble proteins can provide significant insight into transmembrane protein structures. A complementary novel two-stage modeling/selection process that mimics the two-stage helical membrane protein folding was developed. Combined with the scoring function, the method was successfully applied to model 5 transmembrane proteins. The root mean square deviations of the predicted models ranged from 5.0 to 8.8 Å to the native structures.


Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Biología Computacional , Bases de Datos de Proteínas , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Soluciones/química
18.
Curr Opin Biotechnol ; 23(4): 545-54, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22321941

RESUMEN

The bacterial flagellar motor (BFM) is a self-assembling rotary nanomachine. It converts a flux of cations into the mechanical rotation of long filaments that propel bacteria through viscous media. The BFM contains a torque-generating ring that is complete with molecular machinery known as the switch complex that allows it to reverse directions. With four billion years of optimization, the BFM probably offers the pinnacle of sophisticated nanorotor design. Moreover as one of the best-characterized large biomolecular complexes, it offers the potential for convergence between nanotechnology and biology, which requires an atomic level understanding of BFM structure and function. This review focuses on current molecular models of the reversible BFM and the strategies used to derive them.


Asunto(s)
Proteínas Bacterianas/química , Flagelos/química , Modelos Moleculares , Proteínas Motoras Moleculares/química , Salmonella/química , Rotación , Salmonella/metabolismo , Torque
19.
Nat Commun ; 3: 687, 2012 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-22353718

RESUMEN

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases.


Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Thermus thermophilus/enzimología , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Thermus thermophilus/citología
20.
Nat Struct Mol Biol ; 17(3): 373-8, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20173764

RESUMEN

Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23 A resolution EM density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases.


Asunto(s)
Modelos Moleculares , ATPasas de Translocación de Protón/química , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
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