RESUMO
Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments.
Assuntos
Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Interferometria , Bicamadas Lipídicas/química , Potenciais da Membrana , Simulação de Dinâmica Molecular , Nêutrons , Domínios ProteicosRESUMO
The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD's profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure.
Assuntos
Interferometria/métodos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Nêutrons , Raios X , Bicamadas Lipídicas/química , Fosfolipídeos/químicaRESUMO
This work reports the first example of a single-chain protein computationally designed to contain four α-helical segments and fold to form a four-helix bundle encapsulating a supramolecular abiological chromophore that possesses exceptional nonlinear optical properties. The 109-residue protein, designated SCRPZ-1, binds and disperses an insoluble hyperpolarizable chromophore, ruthenium(II) [5-(4'-ethynyl-(2,2';6',2â³-terpyridinyl))-10,20-bis(phenyl)porphinato]zinc(II)-(2,2';6',2â³-terpyridine)(2+) (RuPZn) in aqueous buffer solution at a 1:1 stoichiometry. A 1:1 binding stoichiometry of the holoprotein is supported by electronic absorption and circular dichroism spectra, as well as equilibrium analytical ultracentrifugation and size exclusion chromatography. SCRPZ-1 readily dimerizes at micromolar concentrations, and an empirical redesign of the protein exterior produced a stable monomeric protein, SCRPZ-2, that also displayed a 1:1 protein:cofactor stoichiometry. For both proteins in aqueous buffer, the encapsulated cofactor displays photophysical properties resembling those exhibited by the dilute RuPZn cofactor in organic solvent: femtosecond, nanosecond, and microsecond time scale pump-probe transient absorption spectroscopic data evince intensely absorbing holoprotein excited states having large spectral bandwidth that penetrate deep in the near-infrared energy regime; the holoprotein electronically excited triplet state exhibits a microsecond time scale lifetime characteristic of the RuPZn chromophore. Hyper-Rayleigh light scattering measurements carried out at an incident irradiation wavelength of 1340 nm for these holoproteins demonstrate an exceptional dynamic hyperpolarizabilty (ß1340 = 3100 × 10(-30) esu). X-ray reflectivity measurements establish that this de novo-designed hyperpolarizable protein can be covalently attached with high surface density to a silicon surface without loss of the cofactor, indicating that these assemblies provide a new approach to bioinspired materials that have unique electro-optic functionality.
Assuntos
Simulação por Computador , Metaloporfirinas/química , Proteínas/química , Sequência de Aminoácidos , Dicroísmo Circular , Desenho de Fármacos , Metaloporfirinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas/genética , Especificidade por SubstratoRESUMO
We show that simply designed amphiphilic 4-helix bundle peptides can be utilized to vectorially orient a linearly extended donor-bridge-acceptor (D-br-A) electron transfer (ET) chromophore within its core. The bundle's interior is shown to provide a unique solvation environment for the D-br-A assembly not accessible in conventional solvents and thereby control the magnitudes of both light-induced ET and thermal charge recombination rate constants. The amphiphilicity of the bundle's exterior was employed to vectorially orient the peptide-chromophore complex at a liquid-gas interface, and its ends were tailored for subsequent covalent attachment to an inorganic surface, via a "directed assembly" approach. Structural data, combined with evaluation of the excited state dynamics exhibited by these peptide-chromophore complexes, demonstrate that densely packed, acentrically ordered 2-D monolayer ensembles of such complexes at high in-plane chromophore densities approaching 1/200 Å(2) offer unique potential as active layers in binary heterojunction photovoltaic devices.
Assuntos
Nanoestruturas/química , Peptídeos/química , Transporte de Elétrons , Estrutura Secundária de ProteínaRESUMO
"Push-pull" chromophores based on extended pi-electron systems have been designed to exhibit exceptionally large molecular hyperpolarizabilities. We have engineered an amphiphilic four-helix bundle peptide to vectorially incorporate such hyperpolarizable chromophores having a metalloporphyrin moiety, with high specificity into the interior core of the bundle. The amphiphilic exterior of the bundle facilitates the formation of densely packed monolayer ensembles of the vectorially oriented peptide-chromophore complexes at the liquid-gas interface. Chemical specificity designed into the ends of the bundle facilitates the subsequent covalent attachment of these monolayer ensembles onto the surface of an inorganic substrate. In this article, we describe the structural characterization of these monolayer ensembles at each stage of their fabrication for one such peptide-chromophore complex designated as AP0-RuPZn. In the accompanying article, we describe the characterization of their macroscopic nonlinear optical properties.
Assuntos
Metaloporfirinas/química , Dinâmica não Linear , Fenômenos Ópticos , Compostos Organometálicos/química , Peptídeos/química , Sequência de Aminoácidos , Gases/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Água/química , Difração de Raios XRESUMO
The macroscopic nonlinear optical response of the "push-pull" chromophore RuPZn incorporated into a single monolayer of the amphiphilic 4-helix bundle peptide (AP0) covalently attached to a solid substrate at high in-plane density has been measured. The second-order susceptibility, chi(zzz), was found to be in the range of approximately 15 x 10(-9) esu, consistent with a coherent sum of the nonlinear contributions from the individual chromophores (beta) as previously measured in isotropic solution through hyper-Rayleigh scattering as well as estimated from theoretical calculations. The microscopic hyperpolarizability of the RuPZn chromophore is preserved upon incorporation into the peptide monolayer, suggesting that the chromophore-chromophore interactions in the densely packed ensemble do not substantially affect the first-order molecular hyperpolarizability. The polarization angle dependence of the second harmonic signal reveals that the chromophore is vectorially oriented in the two-dimensional ensemble. Analysis of the order parameter together with information obtained from grazing incidence X-ray diffraction help in determining the chromophore orientation within the AP0-RuPZn monolayer. Taking into account an average pitch angle of approximately 20 degrees characterizing the coiled-coil structure of the peptide bundle, the width of the bundle's tilt angle distribution should be sigma < or = 20 degrees, resulting in a mean value of the tilt angle 23 degrees < or = theta(0) < or = 37 degrees.
Assuntos
Compostos de Rutênio/química , Análise dos Mínimos Quadrados , Difração de Raios XRESUMO
The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.
Assuntos
Proteínas de Membrana/química , Complexos Multiproteicos/química , Porfirinas/química , Dicroísmo Circular , Modelos Moleculares , Ligação Proteica , Dobramento de ProteínaRESUMO
A nitrile-derived amino acid, Phe(CN), has been used as an internal spectroscopic probe to study the binding of an inhalational anesthetic to a model membrane protein. The infrared spectra from experiment showed a blue-shift of the nitrile vibrational frequency in the presence of the anesthetic halothane. To interpret the infrared results and explore the nature of the interaction between halothane and the model protein, all-atom molecular dynamics (MD) simulations have been used to probe the structural and dynamic properties of the protein in the presence and absence of one halothane molecule. The frequency shift analyzed from MD simulations agrees well with the experimental infrared results. Decomposition of the forces acting on the nitrile probes demonstrates an indirect impact on the probes from halothane, namely a change of the protein's electrostatic local environment around the probes induced by halothane. Although the halothane remains localized within the designed hydrophobic binding cavity, it undergoes a significant amount of translational and rotational motion, modulated by the interaction of the trifluorine end of halothane with backbone hydrogens of the residues forming the cavity. This dominant interaction between halothane and backbone hydrogens outweighs the direct interaction between halothane and the nitrile groups, making it a good "spectator" probe of the halothane-protein interaction. These MD simulations provide insight into action of anesthetic molecules on the model membrane protein, and also support the further development of nitrile-labeled amino acids as spectroscopic probes within the designed binding cavity.
Assuntos
Alanina/análogos & derivados , Anestésicos Gerais/química , Corantes Fluorescentes/química , Halotano/química , Canais Iônicos/química , Modelos Moleculares , Nitrilas/química , Alanina/química , Anestésicos Gerais/metabolismo , Anestésicos Gerais/farmacologia , Halotano/metabolismo , Halotano/farmacologia , Hidrogênio/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Conformação Molecular , Movimento , Mutação , Ligação Proteica , Espectrofotometria Infravermelho , VibraçãoRESUMO
We demonstrate that cyano-phenylalanine (Phe(CN)) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-Phe(CN). The Trp to Phe(CN) mutation alters neither the alpha-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this Phe(CN) mutant hbAP-Phe(CN), based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-Phe(CN), enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four Phe(CN) probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-Phe(CN) therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.
Assuntos
Alanina/análogos & derivados , Anestésicos Gerais/química , Corantes Fluorescentes/química , Halotano/química , Canais Iônicos/química , Nitrilas/química , Vibração , Ar , Alanina/química , Sequência de Aminoácidos , Anestésicos Gerais/metabolismo , Soluções Tampão , Dicroísmo Circular , Detergentes/química , Halotano/metabolismo , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Infravermelho , Propriedades de Superfície , Água/química , Raios XRESUMO
We previously reported the synthesis and structural characterization of a model membrane protein comprised of an amphiphilic 4-helix bundle peptide with a hydrophobic domain based on a synthetic ion channel and a hydrophilic domain with designed cavities for binding the general anesthetic halothane. In this work, we synthesized an improved version of this halothane-binding amphiphilic peptide with only a single cavity and an otherwise identical control peptide with no such cavity, and applied x-ray reflectivity to monolayers of these peptides to probe the distribution of halothane along the length of the core of the 4-helix bundle as a function of the concentration of halothane. At the moderate concentrations achieved in this study, approximately three molecules of halothane were found to be localized within a broad symmetric unimodal distribution centered about the designed cavity. At the lowest concentration achieved, of approximately one molecule per bundle, the halothane distribution became narrower and more peaked due to a component of approximately 19A width centered about the designed cavity. At higher concentrations, approximately six to seven molecules were found to be uniformly distributed along the length of the bundle, corresponding to approximately one molecule per heptad. Monolayers of the control peptide showed only the latter behavior, namely a uniform distribution along the length of the bundle irrespective of the halothane concentration over this range. The results provide insight into the nature of such weak binding when the dissociation constant is in the mM regime, relevant for clinical applications of anesthesia. They also demonstrate the suitability of both the model system and the experimental technique for additional work on the mechanism of general anesthesia, some of it presented in the companion parts II and III under this title.
Assuntos
Anestésicos Gerais/química , Halotano/química , Canais Iônicos/química , Sequência de Aminoácidos , Anestésicos Gerais/metabolismo , Halotano/metabolismo , Canais Iônicos/síntese química , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Raios XRESUMO
We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.
Assuntos
Metaloporfirinas/química , Nanoestruturas/química , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Compostos Férricos/síntese química , Compostos Férricos/química , Metaloporfirinas/síntese química , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
Incorporation of extended conjugated chromophores into amphiphilic 4-helix bundle peptides has been shown experimentally to be an effective means to orient the chromphores vectorially in 2-D ensembles with high in-plane density. The designed microscopic hyperpolarizabilty of the chromophore is preserved in the macroscopic NLO response of the ensemble. We show via molecular dynamics simulation that the designed coiled-coil structure of the bundle controls the conformation and dynamics of the chromophore that are critical to optimizing its hyperpolarizability.
Assuntos
Corantes/química , Simulação por Computador , Ligadura , Metaloporfirinas/química , Modelos Químicos , Conformação Molecular , Peptídeos/química , Conformação Proteica , Piridinas/químicaRESUMO
The mechanism of electromechanical coupling for voltage-gated ion channels (VGICs) involved in neurological signal transmission, primarily Nav- and Kv-channels, remains unresolved. Anesthetics have been shown to directly impact this mechanism, at least for Kv-channels. Molecular dynamics computer simulations can now predict the structures of VGICs embedded within a hydrated phospholipid bilayer membrane as a function of the applied transmembrane voltage, but significant assumptions are still necessary. Nevertheless, these simulations are providing new insights into the mechanism of electromechanical coupling at the atomic level in 3-D. We show that time-resolved neutron interferometry can be used to investigate directly the profile structure of a VGIC, vectorially oriented within a single hydrated phospholipid bilayer membrane at the solid-liquid interface, as a function of the applied transmembrane voltage in the absence of any assumptions or potentially perturbing modifications of the VGIC protein and/or the host membrane. The profile structure is a projection of the membrane's 3-D structure onto the membrane normal and, in the absence of site-directed deuterium labeling, is provided at substantially lower spatial resolution than the atomic level. Nevertheless, this novel approach can be used to directly test the validity of the predictions from molecular dynamics simulations. We describe the key elements of our novel experimental approach, including why each is necessary and important to providing the essential information required for this critical comparison of "simulation" vs "experiment." In principle, the approach could be extended to higher spatial resolution and to include the effects of anesthetics on the electromechanical coupling mechanism in VGICs.
Assuntos
Anestésicos/química , Medição da Troca de Deutério/métodos , Interferometria/métodos , Bicamadas Lipídicas/química , Nêutrons , Fosfatidilcolinas/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Anestésicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Interferometria/instrumentação , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilcolinas/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Raios XRESUMO
The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle peptides (or "maquettes"), capable of binding metallo-porphyrin cofactors at selected locations along the length of the core of the bundle, are investigated via molecular dynamics simulations. The rapid evolution of the initial design to stable three-dimensional structures in the absence (apo-form) and presence (holo-form) of bound cofactors is described for the maquettes at two different soft interfaces between polar and nonpolar media. This comparison of the apo- versus holo-forms allows the investigation of the effects of cofactor incorporation on the structure of the four-helix bundle. The simulation results are in qualitative agreement with available experimental data describing the structures at lower resolution and limited dimension.
Assuntos
Simulação por Computador , Metaloporfirinas/química , Peptídeos/química , Proteínas/química , Solventes/química , Sítios de Ligação , Ligação de Hidrogênio , Octanos/química , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Fatores de Tempo , Água/químicaRESUMO
Artificial peptides can be designed to possess a variety of functionalities. If these peptides can be ordered in an ensemble, the functionality can impart macroscopic material properties to the ensemble. Neutron reflectivity is shown to be an effective probe of the intramolecular structures of such peptides vectorially oriented at an interface, key to ensuring that the designed molecular structures translate into the desired material properties of the interface. A model-independent method is utilized to analyze the neutron reflectivity from an alkylated, di- alpha -helical peptide, containing perdeuterated leucine residues at one or two pre-selected positions, in mixed Langmuir monolayers with a phospholipid. The results presented here are more definitive than prior work employing x-ray reflectivity. They show explicitly that the di-helical peptide retains its designed alpha -helical secondary structure at the interface, when oriented perpendicular to the interface at high surface pressure, with the helices projecting into the aqueous subphase without penetrating the layer of phospholipid headgroups.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Difração de Nêutrons/métodos , Fosfolipídeos/química , Proteínas de Membrana/análise , Sondas Moleculares/análise , Sondas Moleculares/química , Conformação Proteica , Espalhamento de RadiaçãoRESUMO
Resonant x-ray reflectivity exploits the energy dependence of atomic scattering factors to locate resonant atoms within the electron density distribution of thin films. We apply the technique to a monolayer of bromo-stearic acid at the air/water interface. The data collection protocol employed cycles through several energies in the vicinity of the bromine K absorption edge and verifies that the energy dependencies observed are indeed resonant effects. The analysis specifies the location of the Br atom with sub-angstrom precision and must consider both the real and imaginary parts of the changes in the scattering factor to be consistent with the known structure and stoichiometry of this test case.
Assuntos
Algoritmos , Bromo/análise , Bromo/química , Ácidos Esteáricos/análise , Ácidos Esteáricos/química , Difração de Raios X/métodos , Ácidos Graxos/análise , Ácidos Graxos/química , Membranas ArtificiaisRESUMO
Langmuir monolayers provide an important system for the investigation of the intramolecular structure and intermolecular ordering of organic and bio-organic macromolecular amphiphiles at an interface between polar and nonpolar media, e.g., the liquid-gas interface. Specular x-ray and neutron reflectivity have contributed substantially to these investigations. However, these reflectivity techniques are generally limited by the absence of crucial phase information, the relatively small contribution of the amphiphile to the scattering-length density contrast across the interface, and the relatively limited range of momentum transfer available perpendicular to the interface. Although several procedures have been developed to provide model-independent solutions to the phase problem, there remains a limited ability to distinguish features of slightly differing contrast (i.e., the "sensitivity") as well as their minimum allowable separation (i.e., the "spatial resolution") along the length of the scattering-length density profile derived from the reflectivity data via solution to the phase problem. Here, we demonstrate how the well-known interferometric approach can be extended to the structural investigation of otherwise unperturbed Langmuir monolayers of these amphiphiles to provide a direct solution to the phase problem and importantly, substantially enhance both the sensitivity and the spatial resolution in the derived profiles.
Assuntos
Gases/química , Interferometria/métodos , Difração de Raios X , Elétrons , Modelos Moleculares , Peptídeos/química , Estrutura Secundária de Proteína , Elementos de Transição/químicaRESUMO
An UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers under in situ conditions is described. The spectrometer utilizes a stand-alone multipass sensor, which is placed in a Langmuir trough and coupled with light source and spectrometer head via fiber optics. Implementation of the multipass scheme in the absorbance sensor makes it possible to obtain reliable quantitative spectroscopic data of the Langmuir monolayers with absorbance as low as 1 mOD. Such high sensitivity makes the developed sensor very useful for UV-visible spectral studies of a wide variety of chromophores. The new technique was applied to several model systems: fatty acid monolayers containing amphiphilic dyes DiI or BODIPY and also a monolayer of a synthetic amphiphilic porphyrin-binding peptide BBC16. Implementation of UV-visible absorbance spectroscopy measurements in situ together with x-ray scattering technique was used to confirm the bound state of the chromophore, and determine the exact position of the latter in the peptide matrix. Fiber optics design of the spectrometer provides portability and compatibility with other experimental techniques making it possible to study samples with a geometry unsuitable for conventional spectroscopic measurements and located in experimental environments with spatial limitations, such as synchrotron x-ray scattering stations.
Assuntos
Dicroísmo Circular/instrumentação , Bicamadas Lipídicas/química , Espectrofotometria Ultravioleta/instrumentação , Ar , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Propriedades de Superfície , Água/químicaRESUMO
We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes.
Assuntos
Metaloproteínas/química , Porfirinas/química , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Metaloproteínas/síntese química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Porfirinas/síntese química , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Termodinâmica , UltracentrifugaçãoRESUMO
Multipigment ensembles that feature (porphinato)metal components and appropriate ethyne- and oligoyne-based chromophore-to-chromophore connectivity can manifest large optical polarizabilities and hyperpolarizabilities by design. Their vectorial orientation and local environment are controlled upon incorporation into designed amphiphilic 4-helix bundle peptides via axial histidyl ligation without disturbing the peptide's helical secondary structure. The chromophore/peptide stoichiometry can be tuned by varying the peptide's oligomeric state. The chromophore/peptide complexes are thermally stable, making them ideal candidates for the fabrication of nonlinear optical biomolecular materials.