RESUMO
Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences. The model always ranks the binding of the cognate sequence in the top 100 sequences, and for 6 of the 8 mAbs, the cognate sequence ranks in the top ten. Practically, this approach has potential utility in selecting highly specific mAbs for therapeutics or diagnostics. More fundamentally, this demonstrates that very sparse random sampling of a large amino acid sequence spaces is sufficient to generate comprehensive models predictive of highly specific molecular recognition.
Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Sequência de Aminoácidos , Aprendizado de Máquina , Epitopos/imunologia , Epitopos/química , Humanos , Ligação Proteica , Sítios de Ligação de Anticorpos , Simulação por ComputadorRESUMO
Past studies have shown that incubation of human serum samples on high density peptide arrays followed by measurement of total antibody bound to each peptide sequence allows detection and discrimination of humoral immune responses to a variety of infectious diseases. This is true even though these arrays consist of peptides with near-random amino acid sequences that were not designed to mimic biological antigens. This "immunosignature" approach, is based on a statistical evaluation of the binding pattern for each sample but it ignores the information contained in the amino acid sequences that the antibodies are binding to. Here, similar array-based antibody profiles are instead used to train a neural network to model the sequence dependence of molecular recognition involved in the immune response of each sample. The binding profiles used resulted from incubating serum from 5 infectious disease cohorts (Hepatitis B and C, Dengue Fever, West Nile Virus and Chagas disease) and an uninfected cohort with 122,926 peptide sequences on an array. These sequences were selected quasi-randomly to represent an even but sparse sample of the entire possible combinatorial sequence space (~1012). This very sparse sampling of combinatorial sequence space was sufficient to capture a statistically accurate representation of the humoral immune response across the entire space. Processing array data using the neural network not only captures the disease-specific sequence-binding information but aggregates binding information with respect to sequence, removing sequence-independent noise and improving the accuracy of array-based classification of disease compared with the raw binding data. Because the neural network model is trained on all samples simultaneously, a highly condensed representation of the differential information between samples resides in the output layer of the model, and the column vectors from this layer can be used to represent each sample for classification or unsupervised clustering applications.
Assuntos
Anticorpos , Doenças Transmissíveis , Humanos , Sequência de Aminoácidos , Peptídeos/química , ImunidadeRESUMO
Photosynthetic organisms organize discrete light-harvesting complexes into large-scale networks to facilitate efficient light collection and utilization. Inspired by nature, herein, synthetic DNA templates were used to direct the formation of dye aggregates with a cyanine dye, K21, into discrete branched photonic complexes, and two-dimensional (2D) excitonic networks. The DNA templates ranged from four-arm DNA tiles, ≈10â nm in each arm, to 2D wireframe DNA origami nanostructures with different geometries and varying dimensions up to 100×100â nm. These DNA-templated dye aggregates presented strongly coupled spectral features and delocalized exciton characteristics, enabling efficient photon collection and energy transfer. Compared to the discrete branched photonic systems templated on individual DNA tiles, the interconnected excitonic networks showed approximately a 2-fold increase in energy transfer efficiency. This bottom-up assembly strategy paves the way to create 2D excitonic systems with complex geometries and engineered energy pathways.
Assuntos
DNA , Nanoestruturas , Transferência de Energia , DNA/química , Nanoestruturas/química , Replicação do DNA , Óptica e FotônicaRESUMO
In combinatorial chemical approaches, optimizing the composition and arrangement of building blocks toward a particular function has been done using a number of methods, including high throughput molecular screening, molecular evolution, and computational prescreening. Here, a different approach is considered that uses sparse measurements of library molecules as the input to a machine learning algorithm which generates a comprehensive, quantitative relationship between covalent molecular structure and function that can then be used to predict the function of any molecule in the possible combinatorial space. To test the feasibility of the approach, a defined combinatorial chemical space consisting of â¼1012 possible linear combinations of 16 different amino acids was used. The binding of a very sparse, but nearly random, sampling of this amino acid sequence space to 9 different protein targets is measured and used to generate a general relationship between peptide sequence and binding for each target. Surprisingly, measuring as little as a few hundred to a few thousand of the â¼1012 possible molecules provides sufficient training to be highly predictive of the binding of the remaining molecules in the combinatorial space. Furthermore, measuring only amino acid sequences that bind weakly to a target allows the accurate prediction of which sequences will bind 10-100 times more strongly. Thus, the molecular recognition information contained in a tiny fraction of molecules in this combinatorial space is sufficient to characterize any set of molecules randomly selected from the entire space, a fact that potentially has significant implications for the design of new chemical function using combinatorial chemical libraries.
Assuntos
Aprendizado de Máquina , Peptídeos/química , Sequência de Aminoácidos , Técnicas de Química Combinatória , Ensaios de Triagem em Larga Escala , Ligantes , Modelos Moleculares , Estrutura Molecular , Biblioteca de Peptídeos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Strongly coupled molecular dye aggregates have unique optoelectronic properties that often resemble those of light harvesting complexes found in Nature. The exciton dynamics in coupled dye aggregates could enhance the long-range transfer of optical excitation energy with high efficiency. In principle, dye aggregates could serve as important components in molecular-scale photonic devices; however, rational design of these coupled dye aggregates with precise control over their organization, interactions, and dynamics remains a challenge. DNA nanotechnology has recently been used to build an excitonic circuit by organizing pseudoisocyanine (PIC) dyes forming J-aggregates on the templates of poly(dA)-poly(dT) DNA duplexes. Here, the excitonic properties of the PIC J-aggregates on DNA are characterized spectroscopically in detail using poly(dA)-poly(dT) tract lengths of 24 and 48 base pairs. The excitonic properties of these DNA templated dye assemblies depend on the length and sequence of the DNA template. The incorporation of a gap of two GC base pairs between two segments of poly(dA)-poly(dT) DNA markedly reduces the delocalization of excitation in the J-aggregates. With a quantum dot (QD) as the light absorber and energy donor and using Alexa Fluor 647 (AF647) as the energy acceptor, with a DNA-templated J-aggregate in between, significant energy transfer from QD to AF647 is observed over a distance far longer than possible without the aggregate bridge. By comparing the efficiency of energy transfer through a continuous J-aggregate with the efficiency when the aggregate has a discontinuity in the middle, the effects of energy transfer within the aggregate bridge between the donor and acceptor are evaluated.
Assuntos
DNA/química , Corantes Fluorescentes/química , Pontos Quânticos/química , Quinolinas/química , Transferência de Energia , Nanotecnologia , Poli A/química , Poli T/químicaRESUMO
The benzothiazole cyanine dye K21 forms dye aggregates on double-stranded DNA (dsDNA) templates. These aggregates exhibit a red-shifted absorption band, enhanced fluorescence emission, and an increased fluorescence lifetime, all indicating strong excitonic coupling among the dye molecules. K21 aggregate formation on dsDNA is only weakly sequence dependent, providing a flexible approach that is adaptable to many different DNA nanostructures. Donor (D)-bridge (B)-acceptor (A) complexes consisting of Alexa Fluor 350 as the donor, a 30 bp (9.7 nm) DNA templated K21 aggregate as the bridge, and Alexa Fluor 555 as the acceptor show an overall donor to acceptor energy transfer efficiency of â¼60%, with the loss of excitation energy being almost exclusively at the donor-bridge junction (63%). There was almost no excitation energy loss due to transfer through the aggregate bridge, and the transfer efficiency from the aggregate to the acceptor was about 96%. By comparing the energy transfer in templated aggregates at several lengths up to 32 nm, the loss of energy per nanometer through the K21 aggregate bridge was determined to be <1%, suggesting that it should be possible to construct structures that use much longer energy transfer "wires" for light-harvesting applications in photonic systems.
Assuntos
Carbocianinas/química , DNA/química , Corantes Fluorescentes/química , Transferência de Energia , Nanoestruturas/químicaRESUMO
Energetics, protein dynamics, and electronic coupling are the key factors in controlling both electron and energy transfer in photosynthetic bacterial reaction centers (RCs). Here, we examine the rates and mechanistic pathways of the P+HA- radical-pair charge recombination, triplet state formation, and subsequent triplet energy transfer from the triplet state of the bacteriochlorophyll dimer (P) to the carotenoid in a series of mutant RCs (L131LH + M160LH (D1), L131LH + M197FH (D2), and L131LH + M160LH + M197FH (T1)) of Rhodobacter sphaeroides. In these mutants, the electronic structure of P is perturbed and the P/P+ midpoint potential is systematically increased due to addition of hydrogen bonds between P and the introduced residues. High-resolution, broad-band, transient absorption spectroscopy on the femtosecond to microsecond timescale shows that the charge recombination rate increases and the triplet energy transfer rate decreases in these mutants relative to the wild type (WT). The increase of the charge recombination rate is correlated to the increase in the energy level of P+HA- and the increase in the P/P+ midpoint potential. On the other hand, the decrease in rate of triplet energy transfer in the mutants can be explained in terms of a lower energy of 3P and a shift in the electron spin density distribution in the bacteriochlorophylls of P. The triplet energy-transfer rate follows the order of WT > L131LH + M197FH > L131LH + M160LH > L131LH + M160LH + M197FH, both at room temperature and at 77 K. A pronounced temperature dependence of the rate is observed for all of the RC samples. The activation energy associated to this process is increased in the mutants relative to WT, consistent with a lower 3P energy due to the addition of hydrogen bonds between P and the introduced residues.
Assuntos
Proteínas de Bactérias/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Proteínas de Bactérias/genética , Carotenoides/química , Transferência de Energia , Ligação de Hidrogênio , Cinética , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Rhodobacter sphaeroides/química , Temperatura , TermodinâmicaRESUMO
Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer. We then use structure-based modelling and quantum dynamics to guide the rational design of higher-order synthetic circuits consisting of multiple discrete dye aggregates within a DX-tile. These programmed circuits exhibit excitonic transport properties with prominent circular dichroism, superradiance, and fast delocalized exciton transfer, consistent with our quantum dynamics predictions. This bottom-up strategy offers a versatile approach to the rational design of strongly coupled excitonic circuits using spatially organized dye aggregates for use in coherent nanoscale energy transport, artificial light-harvesting, and nanophotonics.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Óptica e Fotônica/métodosRESUMO
There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.
Assuntos
Peptídeos/química , Aminoácidos/química , Análise de Sequência/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Three- to four-times higher performance of biohybrid photoelectrochemical cells with photosynthetic reaction centers (RC) has been achieved by using a DNA-based biomimetic antenna. Synthetic dyes Cy3 and Cy5 were chosen and strategically placed in the anntena in such a way that they can collect additional light energy in the visible region of the solar spectrum and transfer it to RC through Förster resonance energy transfer (FRET). The antenna, a DNA templated multiple dye system, is attached to each Rhodobacter sphaeroides RC near the primary donor, P, to facilitate the energy transfer process. Excitation with a broad light spectrum (approximating sunlight) triggers a cascade of excitation energy transfer from Cy3 to Cy5 to P, and also directly from Cy5 to P. This additional excitation energy increases the RC absorbance cross-section in the visible and thus the performance of the photoelectrochemical cells. DNA-based biomimetic antennas offer a tunable, modular light-harvesting system for enhancing RC solar coverage and performance for photoelectrochemical cells.
Assuntos
DNA/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Materiais Biomiméticos , Eletricidade , Transferência de Energia , Corantes Fluorescentes/química , Estrutura Molecular , Fotossíntese , Rhodobacter sphaeroides , Energia Solar , Relação Estrutura-Atividade , Luz SolarRESUMO
In purple bacterial reaction centers, triplet excitation energy transfer occurs from the primary donor P, a bacteriochlorophyll dimer, to a neighboring carotenoid to prevent photodamage from the generation of reactive oxygen species. The BB bacteriochlorophyll molecule that lies between P and the carotenoid on the inactive electron transfer branch is involved in triplet energy transfer between P and the carotenoid. To expand the high-resolution spectral and kinetic information available for describing the mechanism, we investigated the triplet excited state formation and energy transfer pathways in the reaction center of Rhodobacter sphaeroides using pump-probe transient absorption spectroscopy over a broad spectral region on the nanosecond to microsecond time scale at both room temperature and at 77 K. Wild-type reaction centers were compared with a reaction center mutant (M182HL) in which BB is replaced by a bacteriopheophytin (Φ), as well as to reaction centers that lack the carotenoid. In wild-type reaction centers, the triplet energy transfer efficiency from P to the carotenoid was essentially unity at room temperature and at 77 K. However, in the M182HL mutant reaction centers, both the rate and efficiency of triplet energy transfer were decreased at room temperature, and at 77 K, no triplet energy transfer was observed, attributable to a higher triplet state energy of the bacteriopheophytin that replaces bacteriochlorophyll in this mutant. Finally, detailed time-resolved spectral analysis of P, carotenoid, and BB (Φ in the M182HL mutant) reveals that the triplet state of the carotenoid is coupled fairly strongly to the bridging intermediate BB in wild-type and Φ in the M182HL mutant, a fact that is probably responsible for the lack of any obvious intermediate 3BB/3Φ transient formation during triplet energy transfer.
Assuntos
Transferência de Energia , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/química , Cinética , Rhodobacter sphaeroides/metabolismo , TemperaturaRESUMO
Taking inspiration from photosynthetic mechanisms in natural systems, we introduced a light-sensitive photo protective quenching element to an artificial light-harvesting antenna model to control the flow of energy as a function of light intensity excitation. The orange carotenoid protein (OCP) is a nonphotochemical quencher in cyanobacteria: under high-light conditions, the protein undergoes a spectral shift, and by binding to the phycobilisome, it absorbs excess light and dissipates it as heat. By the use of DNA as a scaffold, an antenna system made of organic dyes (Cy3 and Cy5) was constructed, and OCP was assembled on it as a modulated quenching element. By controlling the illumination intensity, it is possible to switch the direction of excitation energy transfer from the donor Cy3 to either of two acceptors. Under low-light conditions, energy is transferred from Cy3 to Cy5, and under intense illumination, energy is partially transferred to OCP as well. These results demonstrate the feasibility of controlling the pathway of energy transfer using light intensity in an engineered light-harvesting system.
Assuntos
Proteínas de Bactérias/química , DNA/química , Nanoestruturas/química , Cianobactérias/química , Transferência de Energia , Fluorescência , Corantes Fluorescentes/química , Conformação de Ácido Nucleico , Fotossíntese , Ficobilissomas/químicaRESUMO
The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called ß-type mutants (in which the HA cofactor is called ßA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (ß mutant), the strong energetic mixing of the P(+)BA(-) and P(+)ßA(-) states (the mixed anion is denoted I(-)) increased the rate of charge recombination between P(+) and I(-) in competition with the I(-) â QA forward reaction. This reduced the overall yield of charge separation forming the P(+)QA(-) state. While the kinetics of the primary electron transfer forming P(+)I(-) were essentially identical in all three ß mutants, the rates of the ßA(-) (I(-)) â QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179-7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or ßA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination).
Assuntos
Bacterioclorofilas/química , Transporte de Elétrons , Feofitinas/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Cinética , LigantesRESUMO
The ability to exchange energy and information between biological and electronic materials is critical in the development of hybrid electronic systems in biomedicine, environmental sensing, and energy applications. While sensor technology has been extensively developed to collect detailed molecular information, less work has been done on systems that can specifically modulate the chemistry of the environment with temporal and spatial control. The bacterial photosynthetic reaction center represents an ideal photonic component of such a system in that it is capable of modifying local chemistry via light-driven redox reactions with quantitative control over reaction rates and has inherent spectroscopic probes for monitoring function. Here a well-characterized model system is presented, consisting of a transparent, porous electrode (antimony-doped tin oxide) which is electrochemically coupled to the reaction center via a cytochrome c molecule. Upon illumination, the reaction center performs the 2-step, 2-electron reduction of a ubiquinone derivative which exchanges with oxidized quinone in solution. Electrons from the electrode then move through the cytochrome to reoxidize the reaction center electron donor. The result is a facile platform for performing redox chemistry that can be optically and electronically controlled in time and space.
Assuntos
Antimônio/química , Eletrodos , Transporte de Elétrons , Complexo de Proteínas do Centro de Reação Fotossintética , Proteobactérias , Compostos de EstanhoRESUMO
It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, QA, may be particularly important in electron transfer involving this cofactor. One can substantially increase the flexibility of this region by removing one of the reaction center subunits, the H-subunit. Even with this large change in structure, photoinduced electron transfer to the quinone still takes place. To evaluate the effect of H-subunit removal on electron transfer to QA, we have compared the kinetics of electron transfer and associated spectral evolution for the LM dimer with that of the intact reaction center complex on picosecond to millisecond time scales. The transient absorption spectra associated with all measured electron transfer reactions are similar, with the exception of a broadening in the QX transition and a blue-shift in the QY transition bands of the special pair of bacteriochlorophylls (P) in the LM dimer. The kinetics of the electron transfer reactions not involving quinones are unaffected. There is, however, a 4-fold decrease in the electron transfer rate from the reduced bacteriopheophytin to QA in the LM dimer compared to the intact reaction center and a similar decrease in the recombination rate of the resulting charge-separated state (P(+)QA(-)). These results are consistent with the concept that the removal of the H-subunit results in increased flexibility in the region around the quinone and an associated shift in the reorganization energy associated with charge separation and recombination.
Assuntos
Proteínas de Bactérias/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Proteínas de Bactérias/química , Bacterioclorofilas/química , Dimerização , Transporte de Elétrons , Elétrons , Cinética , Complexo de Proteínas do Centro de Reação Fotossintética/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Quinonas/química , EspectrofotometriaRESUMO
Cascade reactions drive and regulate a variety of metabolic activities. Efficient coupling of substrate transport between enzymes is important for overall pathway activity and also controls the depletion of intermediate molecules that drive the reaction forward. Here, we assembled a three-enzyme pathway on a series of DNA nanoscaffolds to investigate the dependence of their activities on spatial arrangement. Unlike previous studies, the overall activity of the three-enzyme pathway relied less on inter-enzyme distance and more on the geometric patterns that arranged them within a relatively small range of 10-30â nm. Pathway intermediate detection demonstrated that the assembled enzyme systems quickly depleted the intermediate molecules through efficient reaction coupling.
Assuntos
DNA/química , Enzimas/metabolismo , Nanoestruturas/química , Carboxiliases/química , Carboxiliases/metabolismo , DNA/metabolismo , Enzimas/química , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Oxirredução , Especificidade por Substrato , TermodinâmicaRESUMO
Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.
Assuntos
DNA/ultraestrutura , Estabilidade Enzimática , Enzimas/metabolismo , Nanoestruturas/ultraestrutura , Catálise , Glucose Oxidase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Lactato Desidrogenases/metabolismo , Malato Desidrogenase/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , beta-Galactosidase/metabolismoRESUMO
Unstructured interactions between proteins and other molecules or surfaces are often described as nonspecific, and have received relatively little attention in terms of their role in biology. However, despite their lack of a specific binding structure, these unstructured interactions can in fact be very selective. The lack of a specific structure for these interactions makes them more difficult to study in a chemically meaningful way, but one approach is statistical, i.e. simply looking at a large number of different ligands and using that to understand the chemistry of binding. Surface-bound peptide arrays are useful in this regard, and have been used as a model previously for this purpose (Wang and Woodbury, 2014). In that study, the binding of several proteins, including ß-galactosidase, to all possible dipeptides, tripeptides and tetrapeptides (using seven selected amino acids) was performed and analyzed in terms of the charge characteristics, hydrophobicity, etc., of the binding interaction. The current work builds upon that study by starting with a representative subset of the tetrapeptides characterized previously and either extending them by adding all possible combinations of one, two and three amino acids, or by concatenating 57 of the previously characterized tetrapeptides to each other in all possible combinations (including order). The extended and concatenated libraries were analyzed by binding either labeled ß-galactosidase to them or by binding a mixture of 10 different labeled proteins of various sizes, hydrophobicities and charge characteristics to the peptide arrays. By comparing the binding signals from the tetrapeptides or amino acid extensions alone to the binding signals from the complete extended or concatenated sequences, it was possible to evaluate the extent to which affinity and specificity of the whole sequence depends on the subsequences that make it up. The conclusion is that while joining two component sequences together can either greatly increase or decrease overall binding and specificity (relative to the component sequences alone), the contribution to the binding affinity and specificity of the individual binding components is strongly dependent on their position in the peptide; component sequences that bind strongly at the C-terminus of the peptide do not necessarily add substantially to binding and specificity when placed at the N-terminus.
Assuntos
Modelos Químicos , Peptídeos/química , Análise Serial de Proteínas , Motivos de Aminoácidos , Ligação ProteicaRESUMO
A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.