RESUMEN
We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.
Asunto(s)
Técnicas Biosensibles , Proteínas de Unión Periplasmáticas , Humanos , Animales , Ratones , Nicotina , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/metabolismo , Ligandos , Sitios de UniónRESUMEN
We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ΔF/F0 vs [nicotine] (δ-slope, 2.7 µM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.
RESUMEN
Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.
Asunto(s)
ADN , Escherichia coli , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos , Células Procariotas/metabolismo , Homología de Secuencia , Genoma BacterianoRESUMEN
The inaugural "Microbiome for Mars" virtual workshop took place on July 13, 2020. This event assembled leaders in microbiome research and development to discuss their work and how it may relate to long-duration human space travel. The conference focused on surveying current microbiome research, future endeavors, and how this growing field could broadly impact human health and space exploration. This report summarizes each speaker's presentation in the order presented at the workshop.
Asunto(s)
Astronautas , Atención a la Salud/tendencias , Marte , Microbiota/fisiología , Vuelo Espacial , Animales , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiología , Humanos , Microbiota/genéticaRESUMEN
Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.
Asunto(s)
Hemiterpenos/metabolismo , Hidroliasas/genética , Hidroliasas/metabolismo , Cetoácidos/metabolismo , Ingeniería de Proteínas , Valeratos/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Diseño Asistido por Computadora , Hidroliasas/química , Modelos Moleculares , Mutación , Fosforilación , Conformación Proteica , Especificidad por SustratoRESUMEN
The accurate prediction of protein stability upon sequence mutation is an important but unsolved challenge in protein engineering. Large mutational datasets are required to train computational predictors, but traditional methods for collecting stability data are either low-throughput or measure protein stability indirectly. Here, we develop an automated method to generate thermodynamic stability data for nearly every single mutant in a small 56-residue protein. Analysis reveals that most single mutants have a neutral effect on stability, mutational sensitivity is largely governed by residue burial, and unexpectedly, hydrophobics are the best tolerated amino acid type. Correlating the output of various stability-prediction algorithms against our data shows that nearly all perform better on boundary and surface positions than for those in the core and are better at predicting large-to-small mutations than small-to-large ones. We show that the most stable variants in the single-mutant landscape are better identified using combinations of 2 prediction algorithms and including more algorithms can provide diminishing returns. In most cases, poor in silico predictions were tied to compositional differences between the data being analyzed and the datasets used to train the algorithm. Finally, we find that strategies to extract stabilities from high-throughput fitness data such as deep mutational scanning are promising and that data produced by these methods may be applicable toward training future stability-prediction tools.
Asunto(s)
Mutagénesis/genética , Ingeniería de Proteínas , Estabilidad Proteica , Proteínas/química , Sustitución de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Simulación por Computador , Mutación/genética , Dominios Proteicos/genética , Proteínas/genética , TermodinámicaRESUMEN
Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (CuB) and one ~2 nanometers away in the membrane-bound PmoC subunit (CuC). On the basis of these results, we propose that a monocopper site is able to catalyze methane oxidation in pMMO.
Asunto(s)
Cobre/química , Metano/metabolismo , Metanol/metabolismo , Methylococcus capsulatus/enzimología , Oxigenasas/química , Dominio Catalítico , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Conformación ProteicaRESUMEN
Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem â¼ 600-700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979-982], forms a class of bright, monomeric, far-red FPs.
Asunto(s)
Antozoos/metabolismo , Proteínas Luminiscentes/química , Animales , Antozoos/química , Antozoos/genética , Color , Cristalografía por Rayos X , Fluorescencia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Ingeniería de Proteínas , Proteína Fluorescente RojaRESUMEN
We present ProtaBank, a repository for storing, querying, analyzing, and sharing protein design and engineering data in an actively maintained and updated database. ProtaBank provides a format to describe and compare all types of protein mutational data, spanning a wide range of properties and techniques. It features a user-friendly web interface and programming layer that streamlines data deposition and allows for batch input and queries. The database schema design incorporates a standard format for reporting protein sequences and experimental data that facilitates comparison of results across different data sets. A suite of analysis and visualization tools are provided to facilitate discovery, to guide future designs, and to benchmark and train new predictive tools and algorithms. ProtaBank will provide a valuable resource to the protein engineering community by storing and safeguarding newly generated data, allowing for fast searching and identification of relevant data from the existing literature, and exploring correlations between disparate data sets. ProtaBank invites researchers to contribute data to the database to make it accessible for search and analysis. ProtaBank is available at https://protabank.org.
Asunto(s)
Bases de Datos de Proteínas , Proteínas , Algoritmos , Proteínas/químicaRESUMEN
The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).
Asunto(s)
NADP/genética , NAD/genética , Oxidorreductasas/genética , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol/genética , Aldehído Reductasa/genética , Conformación Proteica , Ingeniería de Proteínas/métodos , Especificidad por SustratoRESUMEN
The pursuit of circuits and metabolic pathways of increasing complexity and robustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription-translation (TX-TL) system for responsiveness to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. Promising candidates from the in vitro screen were further characterized in vitro and in vivo in a gene circuit. The screen yielded two qacR mutants that respond to vanillin both in vitro and in vivo. While the mutants exhibit some toxicity to cells, presumably due to off-target effects, they are prime starting points for directed evolution toward vanillin sensors with the specifications required for use in a dynamic control loop. We believe this process, a combination of the generation of variants coupled with in vitro screening, can serve as a framework for designing new sensors for other target compounds.
Asunto(s)
Proteínas Bacterianas/metabolismo , Benzaldehídos/metabolismo , Ingeniería de Proteínas , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistema Libre de Células , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagénesis , Plásmidos/genética , Plásmidos/metabolismo , Biosíntesis de Proteínas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transcripción GenéticaRESUMEN
Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL(-1) were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4 . Scale-up reactions were achieved from 15 to 100 µL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs.
Asunto(s)
Sulfato de Cobre/química , Escherichia coli/genética , Oxidorreductasas/biosíntesis , Sistema Libre de Células , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas/aislamiento & purificación , Biosíntesis de Proteínas , Biología Sintética/métodos , Transcripción GenéticaRESUMEN
Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.
Asunto(s)
Simulación por Computador , ADN/química , Diseño de Fármacos , Nanocables/química , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Proteínas de Drosophila , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Modelos Moleculares , Nanotecnología , Multimerización de Proteína , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein-protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by ß-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix-mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability.
Asunto(s)
Dimerización , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Drosophila , Proteínas de Homeodominio/química , Calor , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Mapeo de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Soluciones , Factores de Transcripción/químicaRESUMEN
Computational design has been used with mixed success for the design of protein surfaces, with directed evolution heretofore providing better practical solutions than explicit design. Directed evolution, however, requires a tractable high-throughput screen because the random nature of mutation does not enrich for desired traits. Here we demonstrate the successful design of the ß-sheet surface of a red fluorescent protein (RFP), enabling control over its oligomerization. To isolate the problem of surface design, we created a hybrid RFP from DsRed and mCherry with a stabilized protein core that allows for monomerization without loss of fluorescence. We designed an explicit library for which 93 of 96 (97%) of the protein variants are soluble, stably fluorescent, and monomeric. RFPs are heavily used in biology, but are natively tetrameric, and creating RFP monomers has proven extremely difficult. We show that surface design and core engineering are separate problems in RFP development and that the next generation of RFP markers will depend on improved methods for core design.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas Luminiscentes/genética , Ingeniería de Proteínas/métodos , Biología Computacional , Fluorescencia , Colorantes Fluorescentes/síntesis química , Biblioteca de Genes , Proteínas Luminiscentes/química , Plásmidos/genética , Conformación Proteica , Multimerización de Proteína/genética , Multimerización de Proteína/fisiología , Proteína Fluorescente RojaRESUMEN
In standard implementations of computational protein design, a positive-design approach is used to predict sequences that will be stable on a given backbone structure. Possible competing states are typically not considered, primarily because appropriate structural models are not available. One potential competing state, the domain-swapped dimer, is especially compelling because it is often nearly identical with its monomeric counterpart, differing by just a few mutations in a hinge region. Molecular dynamics (MD) simulations provide a computational method to sample different conformational states of a structure. Here, we tested whether MD simulations could be used as a post-design screening tool to identify sequence mutations leading to domain-swapped dimers. We hypothesized that a successful computationally designed sequence would have backbone structure and dynamics characteristics similar to that of the input structure and that, in contrast, domain-swapped dimers would exhibit increased backbone flexibility and/or altered structure in the hinge-loop region to accommodate the large conformational change required for domain swapping. While attempting to engineer a homodimer from a 51-amino-acid fragment of the monomeric protein engrailed homeodomain (ENH), we had instead generated a domain-swapped dimer (ENH_DsD). MD simulations on these proteins showed increased B-factors derived from MD simulation in the hinge loop of the ENH_DsD domain-swapped dimer relative to monomeric ENH. Two point mutants of ENH_DsD designed to recover the monomeric fold were then tested with an MD simulation protocol. The MD simulations suggested that one of these mutants would adopt the target monomeric structure, which was subsequently confirmed by X-ray crystallography.
Asunto(s)
Proteínas de Drosophila/ultraestructura , Proteínas de Homeodominio/química , Proteínas de Homeodominio/ultraestructura , Ingeniería de Proteínas/métodos , Isoformas de Proteínas/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/ultraestructura , Animales , Cristalografía por Rayos X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodominio/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas/fisiología , Isoformas de Proteínas/genética , Factores de Transcripción/genéticaRESUMEN
Engineering fluorescent proteins (FPs) to emit light at longer wavelengths is a significant focus in the development of the next generation of fluorescent biomarkers, as far-red light penetrates tissue with minimal absorption, allowing better imaging inside of biological hosts. Structure-guided design and directed evolution have led to the discovery of red FPs with significant bathochromic shifts to their emission. Here, we present the crystal structure of one of the most bathochromically shifted FPs reported to date, AQ143, a nine-point mutant of aeCP597, a chromoprotein from Actinia equina. The 2.19 Å resolution structure reveals several important chromophore interactions that contribute to the protein's far-red emission and shows dual occupancy of the green and red chromophores.
Asunto(s)
Proteínas Luminiscentes/química , Animales , Cristalografía por Rayos X , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/aislamiento & purificación , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Ingeniería de Proteínas , Anémonas de Mar , Proteína Fluorescente RojaRESUMEN
Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.
Asunto(s)
Biocatálisis , Evolución Molecular Dirigida , Enzimas/química , Enzimas/metabolismo , Ingeniería de Proteínas , Carbono/química , Dominio Catalítico , Cristalografía por Rayos X , Enzimas/genética , Cinética , Modelos Moleculares , Protones , Triazoles/química , Triazoles/metabolismo , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID. It takes a three-dimensional protein structure as input and, using an all-atom force field, attempts to improve the energy of the structure by systematically perturbing backbone dihedrals and side-chain rotamer conformations. We compare GRID to Backrub, a stochastic algorithm that has been shown to predict a significant fraction of the conformational changes that occur with point mutations. We applied GRID and Backrub to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. GRID resulted in energy improvements that were significantly better than those attained by Backrub while expending about the same amount of computational resources. GRID resulted in relaxed structures that had slightly higher backbone RMSDs compared to Backrub relative to the starting crystal structures. The average RMSD was 0.25 ± 0.02 Å for GRID versus 0.14 ± 0.04 Å for Backrub. These relatively minor deviations indicate that both algorithms generate structures that retain their original topologies, as expected given the nature of the algorithms.