Structural Prediction of Protein–Protein Interactions by Docking: Application to Biomedical Problems

A huge amount of genetic information is available thanks to the recent advances in sequencing technologies and the larger computational capabilities, but the interpretation of such genetic data at phenotypic level remains elusive. One of the reasons is that proteins are not acting alone, but are specifically interacting with other proteins and biomolecules, forming intricate interaction networks that are essential for the majority of cell processes and pathological conditions. Thus, characterizing such interaction networks is an important step in understanding how information flows from gene to phenotype. Indeed, structural characterization of protein–protein interactions at atomic resolution has many applications in biomedicine, from diagnosis and vaccine design, to drug discovery. However, despite the advances of experimental structural determination, the number of interactions for which there is available structural data is still very small. In this context, a complementary approach is computational modeling of protein interactions by docking, which is usually composed of two major phases: (i) sampling of the possible binding modes between the interacting molecules and (ii) scoring for the identification of the correct orientations. In addition, prediction of interface and hot-spot residues is very useful in order to guide and interpret mutagenesis experiments, as well as to understand functional and mechanistic aspects of the interaction. Computational docking is already being applied to specific biomedical problems within the context of personalized medicine, for instance, helping to interpret pathological mutations involved in protein–protein interactions, or providing modeled structural data for drug discovery targeting protein–protein interactions.Spanish Ministry of Economy grant number BIO2016-79960-R; D.B.B. is supported by a predoctoral fellowship from CONACyT; M.R. is supported by an FPI fellowship from the Severo Ochoa program. We are grateful to the Joint BSC-CRG-IRB Programme in Computational Biology.Peer ReviewedPostprint (author's final draft

Towards a non-equilibrium thermodynamic theory of ecosystem assembly and development

Non-equilibrium thermodynamics has had a significant historic influence on the development of theoretical ecology, even informing the very concept of an ecosystem. Much of this influence has manifested as proposed extremal principles. These principles hold that systems will tend to maximise certain thermodynamic quantities, subject to the other constraints they operate under. A particularly notable extremal principle is the maximum entropy production principle (MaxEPP); that systems maximise their rate of entropy production. However, these principles are not robustly based in physical theory, and suffer from treating complex ecosystems in an extremely coarse manner. To address this gap, this thesis derives a limited but physically justified extremal principle, as well as carrying out a detailed investigation of the impact of non-equilibrium thermodynamic constraints on the assembly of microbial communities. The extremal principle we obtain pertains to the switching between states in simple bistable systems, with switching paths that generate more entropy being favoured. Our detailed investigation into microbial communities involved developing a novel thermodynamic microbial community model, using which we found the rate of ecosystem development to be set by the availability of free-energy. Further investigation was carried out using this model, demonstrating the way that trade-offs emerging from fundamental thermodynamic constraints impact the dynamics of assembling microbial communities. Taken together our results demonstrate that theory can be developed from non-equilibrium thermodynamics, that is both ecologically relevant and physically well grounded. We find that broad extremal principles are unlikely to be obtained, absent significant advances in the field of stochastic thermodynamics, limiting their applicability to ecology. However, we find that detailed consideration of the non-equilibrium thermodynamic mechanisms that impact microbial communities can broaden our understanding of their assembly and functioning.Open Acces

Improving The Usage Of Unnatural Amino Acids In Proteins: Thioamides And Other Biophysical Probes

Methods for genetically and synthetically manipulating protein composition enable a greater flexibility in the study of protein dynamics and function. However, current techniques for incorporating biophysical probes in the form of unnatural amino acids (Uaas) can suffer from poor yield, limited selectivity for the desired probe, and an insufficient understanding of the impact that the probe has on protein structure and function. Each of the studies discussed herein addresses one or more of these shortcomings in an effort to improve the usage of Uaas in biochemistry. We have shown that using inteins as traceless, cleavable purification tags enables the separation of full length Uaa containing proteins from their corresponding truncation products. This method has been used to incorporate Uaas in previously unattainable positions in a variety of proteins using a myriad of Uaas, inteins, and purification tags. In other applications, we have used E. coli aminoacyl transferase (AaT) to selectively modify the N-termini of proteins with Uaas to be used in native chemical ligation or “click” chemistry reactions. Finally, we have previously used backbone thioamide modifications to enable biophysical studies of proteins by taking advantage of their properties as fluorescence quenchers. However, the impact of thioamides on the stability of proteins rich in secondary and tertiary structure has yet to be understood in detail. In this work, we have conducted the most comprehensive studies to date on the effect of thioamides on the structure and thermostability of the full-length proteins, using calmodulin and the B1 domain of protein G. We have found that the thioamide can have destabilizing, neutral, or even stabilizing effects depending on the position of substitution within alpha-helical and beta-sheet folds. Moreover, the advances we have made in thioamide peptide synthesis and protein ligation will enable us to install thioamides with increased efficiency, permitting the first syntheses of proteins with multiple thioamides. In general, by working at the interface of several protein modification technologies, we have developed rigorous methodologies for the incorporation of side chain and backbone modifications while scrutinizing the effects that these modifications may have on protein structure and stability

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery

Non-equilibrium dynamics of actively-driven viscoelastic networks

To maintain internal organization, living systems need to dissipate energy at the molecular level, thus operating far from thermodynamic equilibrium. At the larger scales, non-equilibrium behavior can be manifest through circulation in the phase space of mesoscopic coordinates and various techniques and measures have been developed to detect and quantify this circulation. It is however still not clear what these measures teach us about the physical properties of the system and how they can be employed to make useful predictions. In the following thesis, we will first review recent progress in detecting and quantifying mesoscopic currents in soft living systems; we will then employ minimal models of actively driven viscoelastic networks to understand how the non-equilibrium dynamics are affected by the internal mechanical structure. Finally, we will introduce a method of assessing non-equilibrium fluctuations in a tracking-free fashion via time-lapse microscopy imaging.Um ihre innere Organisation aufrechtzuerhalten, müssen lebende Systeme Energie auf molekularer Ebene dissipieren. Somit arbeiten sie weit entfernt vom thermodynamischen Gleichgewicht. Auf größeren Skalen kann sich Nichtgleichgewichtsverhalten in zirkulärer Bewegung im Phasenraum der mesoskopischen Koordinaten niederschlagen. Um diese Zirkulation zu erkennen und zu quantifizieren, wurden verschiedene Techniken und Methoden entwickelt. Es ist jedoch immer noch nicht klar, was diese Methoden über die physikalischen Eigenschaften des Systems aussagen und wie sie für nützliche Vorhersagen eingesetzt werden können. In dieser Arbeit werden wir zunächst die jüngsten Fortschritte bei der Erkennung und Quantifizierung mesoskopischer Ströme in Systemen aus weicher lebendender Materie untersuchen. Anschließend werden wir minimale Modelle aktiv getriebener viskoelastischer Netzwerke verwenden, um zu verstehen, wie die Nichtgleichgewichtsdynamik durch deren interne mechanische Struktur beeinflusst wird. Schließlich werden wir eine Methode zur Messung von Nichtgleichgewichtsfluktuationen aus Zeitraffermikroskopieaufnahmen, ohne tracking auskommt, einführen

Non-coding RNAs in ovine immunity: Identification of unannotated genes and functional analyses of high throughput genomic data

210 p.Non-coding RNAs (ncRNAs) are involved in several biological processes in mammals, including the immune system response to pathogens and vaccines. The annotation and functional characterization of two of the main classes of ncRNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is more advanced in humans than in livestock species, and thus, there is limited knowledge about the function of these transcripts. The main objective of this work was the identification of ovine non-coding genes, concretely miRNA and lncRNA genes, that are involved in the innate and adaptive immune responses induced by vaccines, vaccine components and pathogen infections. For this purpose, high-throughput transcriptome sequencing datasets produced for this purpose and datasets publicly available were analysed with bioinformatic tools and workflows in order to identify unannotated non-coding genes, profile their expression in different tissues and perform evolutionary conservation analyses. More than 12000 unannotated ovine lncRNAs and 1000 ovine miRNAs were identified in the different analyses, with varying levels of sequence conservation. Differential expression analyses between unstimulated samples and samples stimulated with pathogen infection or vaccination resulted in hundreds of lncRNAs and miRNAs with changed expression. Gene co-expression analyses revealed immune gene-enriched clusters associated with immune system activation. These genes make up a prioritized set of potential candidates for deeper experimental analyses. Taken together, these results should help completing the sheep non-coding gene catalogue, and most importantly, they give evidence of immune state-specific ncRNA expression patterns in a livestock species