RESUMEN
High-quality predicted structures enable structure-based approaches to an expanding number of drug discovery programs. We propose that by utilizing free energy perturbation (FEP), predicted structures can be confidently employed to achieve drug design goals. We use structure-based modeling of hERG inhibition to illustrate this value of FEP.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas , Termodinámica , EntropíaRESUMEN
Biochemistry and molecular biology rely on the recognition of structural complementarity between molecules. Molecular interactions must be both quickly reversible, i.e., tenuous, and specific. How the cell reconciles these conflicting demands is the subject of this article. The problem and its theoretical solution are discussed within the wider theoretical context of the thermodynamics of stochastic processes (stochastic thermodynamics). The solution-an irreversible reaction cycle that decreases internal error at the expense of entropy export into the environment-is shown to be widely employed by biological processes that transmit genetic and regulatory information.
Asunto(s)
Cinética , Procesos Estocásticos , TermodinámicaRESUMEN
We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.
Asunto(s)
Células/citología , Simulación por Computador , Adenosina Trifosfato/metabolismo , Ciclo Celular/genética , Proliferación Celular/genética , Células/metabolismo , Replicación del ADN/genética , Regulación de la Expresión Génica , Imagenología Tridimensional , Cinética , Lípidos/química , Redes y Vías Metabólicas , Metaboloma , Anotación de Secuencia Molecular , Nucleótidos/metabolismo , Termodinámica , Factores de TiempoRESUMEN
Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Evolución Molecular , Código Genético , Selección Genética , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Catálisis , Genotipo , Fenotipo , Filogenia , Biosíntesis de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína , ARN de Transferencia/genética , TermodinámicaRESUMEN
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Familia de Multigenes , Nostoc/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Pollos , Microscopía por Crioelectrón , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Evolución Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestructura , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.
Asunto(s)
Escherichia coli/genética , Chaperonas Moleculares/genética , Pinzas Ópticas , Biosíntesis de Proteínas , Proteoma/química , Ribosomas/genética , Escherichia coli/metabolismo , Humanos , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteoma/biosíntesis , Proteoma/genética , Proteostasis/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , TermodinámicaRESUMEN
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Asunto(s)
Coenzimas/química , Manganeso/química , Oxígeno/química , Complejo de Proteína del Fotosistema II/química , Agua/química , Coenzimas/metabolismo , Cristalografía por Rayos X , Expresión Génica , Rayos Láser , Manganeso/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxígeno/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Teoría Cuántica , Termodinámica , Thermosynechococcus/química , Thermosynechococcus/enzimología , Agua/metabolismoRESUMEN
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a â¼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
Asunto(s)
ADN Glicosilasas/química , ADN Helicasas/química , ADN Polimerasa Dirigida por ADN/química , ADN/química , Endonucleasas/química , Genoma , Proteínas Hierro-Azufre/química , Animales , Bacterias/genética , Bacterias/metabolismo , ADN/metabolismo , ADN/ultraestructura , Daño del ADN , ADN Glicosilasas/metabolismo , ADN Glicosilasas/ultraestructura , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , Espectroscopía de Resonancia por Spin del Electrón , Endonucleasas/metabolismo , Endonucleasas/ultraestructura , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/ultraestructura , Oxidación-Reducción , Unión Proteica , Transducción de Señal , TermodinámicaRESUMEN
Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past.
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Animales , Evolución Molecular Dirigida/métodos , Diseño de Fármacos , Humanos , Modelos Moleculares , Filogenia , Agregado de Proteínas , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Estabilidad Proteica , Proteínas/genética , TermodinámicaRESUMEN
RNAs fold into defined tertiary structures to function in critical biological processes. While quantitative models can predict RNA secondary structure stability, we are still unable to predict the thermodynamic stability of RNA tertiary structure. Here, we probe conformational preferences of diverse RNA two-way junctions to develop a predictive model for the formation of RNA tertiary structure. We quantitatively measured tertiary assembly energetics of >1,000 of RNA junctions inserted in multiple structural scaffolds to generate a "thermodynamic fingerprint" for each junction. Thermodynamic fingerprints enabled comparison of junction conformational preferences, revealing principles for how sequence influences 3-dimensional conformations. Utilizing fingerprints of junctions with known crystal structures, we generated ensembles for related junctions that predicted their thermodynamic effects on assembly formation. This work reveals sequence-structure-energetic relationships in RNA, demonstrates the capacity for diverse compensation strategies within tertiary structures, and provides a path to quantitative modeling of RNA folding energetics based on "ensemble modularity."
Asunto(s)
ARN/metabolismo , Disparidad de Par Base , Biblioteca de Genes , Conformación de Ácido Nucleico , Fotoblanqueo , ARN/química , Pliegue del ARN , Estabilidad del ARN , TermodinámicaRESUMEN
What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.
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Proteínas Bacterianas/química , Hidrolasas/química , Cetosteroides/química , Pseudomonas/enzimología , Esteroide Isomerasas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Expresión Génica , Hidrolasas/genética , Hidrolasas/metabolismo , Cetosteroides/metabolismo , Cinética , Modelos Químicos , Simulación de Dinámica Molecular , Mutación , Pseudomonas/química , Pseudomonas/genética , Espectrofotometría Infrarroja/métodos , Electricidad Estática , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismo , TermodinámicaRESUMEN
Phosphate is essential for all living systems, serving as a building block of genetic and metabolic machinery. However, it is unclear how phosphate could have assumed these central roles on primordial Earth, given its poor geochemical accessibility. We used systems biology approaches to explore the alternative hypothesis that a protometabolism could have emerged prior to the incorporation of phosphate. Surprisingly, we identified a cryptic phosphate-independent core metabolism producible from simple prebiotic compounds. This network is predicted to support the biosynthesis of a broad category of key biomolecules. Its enrichment for enzymes utilizing iron-sulfur clusters, and the fact that thermodynamic bottlenecks are more readily overcome by thioester rather than phosphate couplings, suggest that this network may constitute a "metabolic fossil" of an early phosphate-free nonenzymatic biochemistry. Our results corroborate and expand previous proposals that a putative thioester-based metabolism could have predated the incorporation of phosphate and an RNA-based genetic system. PAPERCLIP.
Asunto(s)
Simulación por Computador , Redes y Vías Metabólicas , Fosfatos/metabolismo , Nucleótidos de Adenina/química , Algoritmos , Coenzima A , Coenzimas , Origen de la Vida , Fosfatos/química , TermodinámicaRESUMEN
The determination of the crystal structures of small-molecule transporters has shed light on the conformational changes that take place during structural isomerization from outward- to inward-facing states. Rather than using a simple rocking movement of two bundles around a central substrate-binding site, it has become clear that even the most simplistic transporters utilize rearrangements of nonrigid bodies. In the most dramatic cases, one bundle is fixed while the other, structurally divergent, bundle carries the substrate some 18 Å across the membrane, which in this review is termed an elevator alternating-access mechanism. Here, we compare and contrast rocker-switch, rocking-bundle, and elevator alternating-access mechanisms to highlight shared features and novel refinements to the basic alternating-access model.
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Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Simulación de Dinámica Molecular , Transporte Biológico , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Expresión Génica , Cinética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
In the last 5 years, most of the molecules that control mitochondrial Ca(2+) homeostasis have been finally identified. Mitochondrial Ca(2+) uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromolecular structure that guarantees Ca(2+) accumulation inside mitochondrial matrix upon increases in cytosolic Ca(2+). Conversely, Ca(2+) release is under the control of the Na(+)/Ca(2+) exchanger, encoded by the NCLX gene, and of a H(+)/Ca(2+) antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca(2+) across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca(2+) homeostasis and the methods used to investigate the dynamics of Ca(2+) concentration within the organelles. We discuss the functional and structural role of the different molecules involved in mitochondrial Ca(2+) handling and their pathophysiological role.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Mitocondrias/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Canales de Calcio/química , Canales de Calcio/genética , Señalización del Calcio , Regulación de la Expresión Génica , Homeostasis , Humanos , Transporte Iónico , Cinética , Mitocondrias/genética , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales , Modelos Moleculares , Intercambiador de Sodio-Calcio/genética , TermodinámicaRESUMEN
The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.
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Receptores ErbB/química , Receptores ErbB/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/aislamiento & purificación , Humanos , Membranas Intracelulares/química , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Termodinámica , Vesículas Transportadoras/químicaRESUMEN
Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Fenómenos Biomecánicos , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Termodinámica , Familia de Proteínas del Síndrome de Wiskott-Aldrich/química , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. Although their mode of action is often compared to that of mechanical machines, a crucial difference is that, at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex-vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and 3D variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.
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Biosíntesis de Proteínas , Ribosomas/química , Ribosomas/ultraestructura , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , ARN de Transferencia/química , Alineación de Secuencia , TermodinámicaRESUMEN
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation ΔF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the ΔF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in ΔF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Mutación , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2-4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2-8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.
Asunto(s)
Proteínas Argonautas/metabolismo , MicroARNs/metabolismo , Animales , Proteínas Argonautas/química , Sitios de Unión , Difusión , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones , Modelos Biológicos , TermodinámicaRESUMEN
Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridizationa small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA.