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MOTIVATION: Engineering high-affinity binders targeting specific antigenic determinants remains a challenging and often daunting task, requiring extensive experimental screening. Computational methods have the potential to accelerate this process, reducing costs and time, but only if they demonstrate broad applicability and efficiency in exploring mutations, evaluating affinity, and pruning unproductive mutation paths. RESULTS: In response to these challenges, we introduce a new computational platform for optimizing protein binders towards their targets. The platform is organized as a series of modules, performing mutation selection and application, molecular dynamics simulations to sample conformations around interaction poses, and mutation prioritization using suitable scoring functions. Notably, the platform supports parallel exploration of different mutation streams, enabling in silico high-throughput screening on High Performance Computing (HPC) systems. Furthermore, the platform is highly customizable, allowing users to implement their own protocols. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://github.com/pgbarletta/locuaz and documentation is at https://locuaz.readthedocs.io/. The data underlying this article are available at https://github.com/pgbarletta/suppl_info_locuaz.
Assuntos
Proteínas , Software , Proteínas/química , Proteínas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Mutação , Biologia Computacional/métodos , Simulação por Computador , Engenharia de Proteínas/métodosRESUMO
The characterization of the statistical ensemble of conformations of intrinsically disordered regions (IDRs) is a great challenge both from experimental and computational points of view. In this respect, a number of protocols have been developed using molecular dynamics (MD) simulations to sample the huge conformational space of the molecule. In this work, we consider one of the best methods available, replica exchange solute tempering (REST), as a reference to compare the results obtained using this method with the results obtained using other methods, in terms of experimentally measurable quantities. Along with the methods assessed, we propose here a novel protocol called probabilistic MD chain growth (PMD-CG), which combines the flexible-meccano and hierarchical chain growth methods with the statistical data obtained from tripeptide MD trajectories as the starting point. The system chosen for testing is a 20-residue region from the C-terminal domain of the p53 tumor suppressor protein (p53-CTD). Our results show that PMD-CG provides an ensemble of conformations extremely quickly, after suitable computation of the conformational pool for all peptide triplets of the IDR sequence. The measurable quantities computed on the ensemble of conformations agree well with those based on the REST conformational ensemble.
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AIM: Evaluate the effectiveness of an intervention to incorporate group and community activities on a sustained basis in all the Basic Health Zones (ZBS) of a health area. DESIGN: During January and February 2019, two members of the research team traveled to each ZBS to interview the medical and nursing coordinators who had previously received an ad hoc initial assessment questionnaire (QAI) by email. PLACE: The scope is the 11 ZBS of a health area. PARTICIPANTS: The study population is the ZBS and the respective teams represented by the medical and nursing coordinations. INTERVENTIONS: Promote a community health commission, carry out training actions, record activities in clinical history and incorporate management objectives. MAIN MEASUREMENTS: Quantitative and qualitative analysis was carried out pre and post after the first year of intervention. RESULTS: In the pre-evaluation: 6 primary care teams (EAP) reported having group activities, 4 were participating in local action projects, 4 had a professional referent for community activities, 3 participated in projects with populations in vulnerable situations and 4 stated have specific meetings on community health. After the intervention: 11 EAPs had group activities, 8 had a reference professional, 6 were participating in local action projects, 4 collaborated in projects with vulnerable populations and 5 held meetings on community health. CONCLUSIONS: The intervention proved effective after its first year of implementation, since all the EAPs carried out group activities and collaborated with the local councils in the area; the majority had leading professionals in community care and, to a lesser extent, participation in local action projects and in vulnerable populations increased.
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Atenção Primária à Saúde , Atenção Primária à Saúde/organização & administração , Humanos , Serviços de Saúde Comunitária/organização & administração , EspanhaRESUMO
The study of proteins with intrinsically disordered regions (IDRs) has emerged as an active field of research due to their intriguing nature. Although IDRs lack a well-defined folded structure, they play important functional roles in cells, following biological mechanisms different from those of the traditional structured proteins. Consequently, it has been necessary to re-design experimental and theoretical methods in order to face the challenges introduced by the dynamic nature of IDRs. In this work, we present an accurate and cost-effective method to study the conformational dynamics of IDRs based on the use of residue-local probabilistic expressions that characterize the conformational ensembles obtained from finite-temperature molecular dynamics (MD) simulations. It is shown that the good performance and the high convergence rates achieved with our method are independent of the IDR lengths, since the method takes advantage of the major influence of the identity and conformation of the nearest residue neighbors on the amino-acid conformational preferences to evaluate the IDR conformational ensembles. This allows us to characterize the conformational space of IDRs using a reduced number of probabilities which can be obtained from comparatively short MD simulations or experimental databases. To exemplify the usefulness of our approach, we present an application to directly detect Molecular Recognition Features (MoRFs) in an IDR domain of the protein p53, and to follow the time evolution of the thermodynamic magnitudes of this system during its exploration of the conformational space.
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Proteínas Intrinsicamente Desordenadas , Conformação Proteica , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Probabilidade , AminoácidosRESUMO
Medicine and technology are constantly evolving. The COVID-19 pandemic has accelerated the development of digitalization in the health sector and specifically of telemedicine. Through a structured bibliographic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) methodology, in this study, the concepts related to telemedicine, its application and the legal regulatory context are defined. With this information, some recommendations and codes of good practice are proposed for their effective implementation in the field of Hepatology.
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COVID-19 , Gastroenterologia , Telemedicina , Humanos , Espanha , Pandemias/prevenção & controleRESUMO
PURPOSE: The mediator (MED) multisubunit-complex modulates the activity of the transcriptional machinery, and genetic defects in different MED subunits (17, 20, 27) have been implicated in neurologic diseases. In this study, we identified a recurrent homozygous variant in MED11 (c.325C>T; p.Arg109Ter) in 7 affected individuals from 5 unrelated families. METHODS: To investigate the genetic cause of the disease, exome or genome sequencing were performed in 5 unrelated families identified via different research networks and Matchmaker Exchange. Deep clinical and brain imaging evaluations were performed by clinical pediatric neurologists and neuroradiologists. The functional effect of the candidate variant on both MED11 RNA and protein was assessed using reverse transcriptase polymerase chain reaction and western blotting using fibroblast cell lines derived from 1 affected individual and controls and through computational approaches. Knockouts in zebrafish were generated using clustered regularly interspaced short palindromic repeats/Cas9. RESULTS: The disease was characterized by microcephaly, profound neurodevelopmental impairment, exaggerated startle response, myoclonic seizures, progressive widespread neurodegeneration, and premature death. Functional studies on patient-derived fibroblasts did not show a loss of protein function but rather disruption of the C-terminal of MED11, likely impairing binding to other MED subunits. A zebrafish knockout model recapitulates key clinical phenotypes. CONCLUSION: Loss of the C-terminal of MED subunit 11 may affect its binding efficiency to other MED subunits, thus implicating the MED-complex stability in brain development and neurodegeneration.
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Complexo Mediador , Microcefalia , Doenças Neurodegenerativas , Animais , Humanos , Homozigoto , Complexo Mediador/genética , Microcefalia/genética , Doenças Neurodegenerativas/genética , RNA , Peixe-Zebra/genéticaRESUMO
Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for.
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Ligantes , Simulação de Dinâmica Molecular , Muramidase/química , Peptídeos/química , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Muramidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de SuperfícieRESUMO
The bottom-up design of smart nanodevices largely depends on the accuracy by which each of the inherent nanometric components can be functionally designed with predictive methods. Here, we present a rationally designed, self-assembled nanochip capable of capturing a target protein by means of pre-selected binding sites. The sensing elements comprise computationally evolved peptides, designed to target an arbitrarily selected binding site on the surface of beta-2-Microglobulin (ß2m), a globular protein that lacks well-defined pockets. The nanopatterned surface was generated by an atomic force microscopy (AFM)-based, tip force-driven nanolithography technique termed nanografting to construct laterally confined self-assembled nanopatches of single stranded (ss)DNA. These were subsequently associated with an ssDNA-peptide conjugate by means of DNA-directed immobilization, therefore allowing control of the peptide's spatial orientation. We characterized the sensitivity of such peptide-containing systems against ß2m in solution by means of AFM-based differential topographic imaging and surface plasmon resonance (SPR) spectroscopy. Our results show that the confined peptides are capable of specifically capturing ß2m from the surface-liquid interface with micromolar affinity, hence providing a viable proof-of-concept for our approach to peptide design.
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Biologia Computacional/métodos , DNA de Cadeia Simples/metabolismo , Peptídeos/metabolismo , Microglobulina beta-2/metabolismo , Sítios de Ligação/genética , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Cinética , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Ressonância de Plasmônio de Superfície/métodos , Microglobulina beta-2/química , Microglobulina beta-2/genéticaRESUMO
OBJECTIVE: To analyze whether the drug safety update issued by the Spanish Agency of Medicines and Healthcare Products (AEMPS), dated October 30, 2018, on agranulocytosis and metamizole contains accurate and necessary information to protect patients from the presentation of this adverse reaction (AR) and if the official documentation of medicines containing metamizole for doctors, pharmacists and the general population conforms to the guidelines of the AEMPS to reduce this risk. SETTING AND PARTICIPANTS: Drug safety update, bibliographic search, information at the European Medicines Agency on metamizole drugs marketed in Spain, technical datasheets, leaflets, Bot PLUS Health Information Database and Catalog of Pharmaceutical Specialties. Notification of 4cases of agranulocytosis due to metamizole after the drug safety update was issued. MAIN INTERVENTIONS AND MEASUREMENTS: Comparison of the key points of the drug safety update and official documents on metamizole with the bibliography. Description of the 4cases of agranulocytosis due to metamizole and application of the causality and severity algorithm. RESULTS: The drug safety update contains omissions and contradiction in respect to the bibliography and the actual use of metamizole in healthcare practice. The official documents show a lack of updating, unapproved indications and doses higher than those recommended. The drug safety update has not stopped the presentation of cases of agranulocytosis due to metamizole. CONCLUSIONS: The AEMPS drug safety update can be improved and it is necessary to update the official information documents on metamizole for health professionals and patients in order to decrease the risk of agranulocytosis.
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Agranulocitose , Dipirona , Agranulocitose/induzido quimicamente , Anti-Inflamatórios não Esteroides/efeitos adversos , Bases de Dados Factuais , Dipirona/efeitos adversos , Humanos , EspanhaRESUMO
The SERS-based detection of protein sequences with single-residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non-aromatic amino acid residues. Herein, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single-molecule detection of 2 similar polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non-aromatic amino acids are detected and discriminated at the single-molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with molecular dynamics simulations, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single-protein analysis, fingerprinting, and sequencing.
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Aminoácidos/química , Peptídeos/química , Análise Espectral Raman/métodos , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Simulação de Dinâmica MolecularRESUMO
Mutation protocols are a key tool in computational biophysics for modelling unknown side chain conformations. In particular, these protocols are used to generate the starting structures for molecular dynamics simulations. The accuracy of the initial side chain and backbone placement is crucial to obtain a stable and quickly converging simulation. In this work, we assessed the performance of several mutation protocols in predicting the most probable conformer observed in finite temperature molecular dynamics simulations for a set of protein-peptide crystals differing only by single-point mutations in the peptide sequence. Our results show that several programs which predict well the crystal conformations fail to predict the most probable finite temperature configuration. Methods relying on backbone-dependent rotamer libraries have, in general, a better performance, but even the best protocol fails in predicting approximately 30% of the mutations.
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Aminoácidos/química , Mutação , Temperatura , Sequência de Aminoácidos , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
Nanobodies offer a viable alternative to antibodies for engineering high affinity binders. Their small size has an additional advantage: it allows exploiting computational protocols for optimizing their biophysical features, such as the binding affinity. The efficient prediction of this quantity is still considered a daunting task especially for modelled complexes. We show how molecular dynamics can successfully assist in the binding affinity prediction of modelled nanobody-protein complexes. The approximate initial configurations obtained by in silico design must undergo large rearrangements before achieving a stable conformation, in which the binding affinity can be meaningfully estimated. The scoring functions developed for the affinity evaluation of crystal structures will provide accurate estimates for modelled binding complexes if the scores are averaged over long finite temperature molecular dynamics simulations.
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Complexo Antígeno-Anticorpo/química , Simulação de Dinâmica Molecular , Proteínas/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/metabolismo , Humanos , Muramidase/química , Muramidase/imunologia , Estrutura Terciária de Proteína , Proteínas/química , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Alinhamento de Sequência , TemperaturaRESUMO
Despite the increasing evidence that conformational transitions in peptides and proteins are driven by specific vibrational energy pathways along the molecule, the current experimental techniques of analysis do as yet not allow to study these biophysical processes in terms of anisotropic energy flows. Computational methods offer a complementary approach to obtain a more detailed understanding of the vibrational and conformational dynamics of these systems. Accordingly, in this work we investigate jointly the vibrational energy distribution and the conformational dynamics of trialanine peptide in water solution at room temperature by applying the Instantaneous Normal Mode analysis to the results derived from equilibrium molecular dynamics simulations. It is shown that conformational changes in trialanine are triggered by the vibrational energy accumulated in the low-frequency modes of the molecule, and that excitation is caused exclusively by thermal fluctuations of the solute-solvent system, thus excluding the possibility of an intramolecular vibrational energy redistribution process.
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Peptídeos/química , Termodinâmica , Modelos Moleculares , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Conformação Proteica , Solventes , Vibração , Água/químicaRESUMO
The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of ß2-microglobulin (ß2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize ß2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design.
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Técnicas de Química Analítica/métodos , Simulação por Computador , Peptídeos Cíclicos/química , Proteínas/isolamento & purificação , Epitopos/química , Modelos Químicos , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/metabolismoRESUMO
The chaperonin complex GroEL-GroES is able to accelerate the folding process of knotted proteins considerably. However, the folding mechanism inside the chaperonin cage is elusive. Here we use a combination of lattice and off-lattice Monte Carlo simulations of simple Go models to study the effect of physical confinement and local flexibility on the folding process of protein model systems embedding a trefoil knot in their native structure. This study predicts that steric confinement plays a specific role in the folding of knotted proteins by increasing the knotting probability for very high degrees of confinement. This effect is observed for protein MJ0366 even above the melting temperature for confinement sizes compatible with the size of the GroEL/GroES chaperonin cage. An enhanced local flexibility produces the same qualitative effects on the folding process. In particular, we observe that knotting probability increases up to 40% in the transition state of protein MJ0366 when flexibility is enhanced. This is underlined by a structural change in the transition state, which becomes devoid of helical content. No relation between the knotting mechanism and flexibility was found in the context of the off-lattice model adopted in this work.
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Proteínas de Bactérias/química , Modelos Moleculares , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Cinética , Método de Monte Carlo , Dobramento de Proteína , Termodinâmica , Temperatura de TransiçãoRESUMO
Using analytical excited-state gradients, vibrational normal modes have been calculated at the minimum of the electronic excited-state potential energy surfaces for a set of extended conjugated molecules with different coupling between them. Molecular model systems composed of units of polyphenylene ethynylene (PPE), polyphenylenevinylene (PPV), and naphthacene/pentacene (NP) have been considered. In all cases except the NP model, the influence of the nonadiabatic coupling on the excited-state equilibrium normal modes is revealed as a unique highest frequency adiabatic vibrational mode that overlaps with the coupling vector. This feature is removed by using a locally diabatic representation in which the effect of NA interaction is removed. Comparison of the original adiabatic modes with a set of vibrational modes computed in the locally diabatic representation demonstrates that the effect of nonadiabaticity is confined to only a few modes. This suggests that the nonadiabatic character of a molecular system may be detected spectroscopically by identifying these unique state-specific high frequency vibrational modes.
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Modelos Moleculares , Análise Espectral/métodos , Vibração , Algoritmos , Naftacenos/química , Polímeros/químicaRESUMO
The folding properties of a protein whose native structure contains a 52 knot are investigated by means of extensive Monte Carlo simulations of a simple lattice model and compared with those of a 31 knot. A 52 knot embedded in the native structure enhances the kinetic stability of the carrier lattice protein in a way that is clearly more pronounced than in the case of the 31 knot. However, this happens at the expense of a severe loss in folding efficiency, an observation that is consistent with the relative abundance of 31 and 52 knots in the Protein Data Bank. The folding mechanism of the 52 knot shares with that of the 31 knot the occurrence of a threading movement of the chain terminus that lays closer to the knotted core. However, co-concomitant knotting and folding in the 52 knot occurs with negligible probability, in sharp contrast to what is observed for the 31 knot. The study of several single point mutations highlights the importance in the folding of knotted proteins of the so-called structural mutations (i.e., energetic perturbations of native interactions between residues that are critical for knotting but not for folding). On the other hand, the present study predicts that mutations that perturb the folding transition state may significantly enhance the kinetic stability of knotted proteins provided they involve residues located within the knotted core.
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Doenças Priônicas/genética , Conformação Proteica , Proteínas/química , Cristalografia por Raios X , Bases de Dados de Proteínas , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Dobramento de Proteína , Proteínas/genética , TermodinâmicaRESUMO
The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.
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Anticorpos , Fragmentos de Imunoglobulinas , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Anticorpos/química , Mutação , Mutagênese , Mutação Puntual , Ligação ProteicaRESUMO
In the framework of the rational design of macromolecules capable of binding to a specific target for biosensing applications, we here further develop an evolutionary protocol designed to optimize the binding affinity of protein binders. In particular we focus on the optimization of the binding portion of small antibody fragments known as nanobodies (or VHH) and choose the hen egg white lysozyme (HEWL) as our target. By implementing a replica exchange scheme for this optimization, we show that an initial hit is not needed and similar solutions can be found by either optimizing an already known anti-HEWL VHH or a randomly selected binder (here a VHH selective towards another macromolecule). While we believe that exhaustive searches of the mutation space are most appropriate when only few key residues have to be optimized, in case a lead binder is not available the proposed evolutionary algorithm should be instead the method of choice.
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Fragmentos de Imunoglobulinas , Anticorpos de Domínio Único , Animais , Fragmentos de Imunoglobulinas/genética , Mutação , Anticorpos de Domínio Único/química , GalinhasRESUMO
Nanobodies (VHHs) are engineered fragments of the camelid single-chain immunoglobulins. The VHH domain contains the highly variable segments responsible for antigen recognition. VHHs can be easily produced as recombinant proteins. Their small size is a good advantage for in silico approaches. Computer methods represent a valuable strategy for the optimization and improvement of their binding affinity. They also allow for epitope selection offering the possibility to design new VHHs for regions of a target protein that are not naturally immunogenic. Here we present an in silico mutagenic protocol developed to improve the binding affinity of nanobodies together with the first step of their in vitro production. The method, already proven successful in improving the low Kd of a nanobody hit obtained by panning, can be employed for the ex novo design of antibody fragments against selected protein target epitopes.