Multi-fractional analysis of molecular diffusion in polymer multilayers by FRAP: a new simulation-based approach

Comprehensive analysis of the multifractional molecular diffusion provides a deeper understanding of the diffusion phenomenon in the fields of material science, molecular and cell biology, advanced biomaterials, etc. Fluorescence recovery after photobleaching (FRAP) is commonly employed to probe the molecular diffusion. Despite FRAP being a very popular method, it is not easy to assess multifractional molecular diffusion due to limited possibilities of approaches for analysis. Here we present a novel simulation-optimization-based approach (S-approach) that significantly broadens possibilities of the analysis. In the S-approach, possible fluorescence recovery scenarios are primarily simulated and afterward compared with a real measurement while optimizing parameters of a model until a sufficient match is achieved. This makes it possible to reveal multifractional molecular diffusion. Fluorescent latex particles of different size and fluorescein isothiocyanate in an aqueous medium were utilized as test systems. Finally, the S-approach has been used to evaluate diffusion of cytochrome c loaded into multilayers made of hyaluronan and polylysine. Software for evaluation of multifractional molecular diffusion by S-approach has been developed aiming to offer maximal versatility and user-friendly way for analysis

An NMR Insight into The Liquid-Liquid Phase Separation of FUS

The formation of two distinct liquid phases from a homogenous solution of solvent and polymer is termed liquid-liquid phase separation (LLPS). The LLPS phenomenon has been described for polymers and proteins. The LLPS of proteins is crucial for the formation of membraneless organelles, which play an important role in intracellular compartmentalization, and is inherently linked to protein fibrillation found in neurodegenerative diseases. The protein fused in sarcoma (FUS) is present in protein fibrils found in amyotrophic lateral sclerosis patients and has been extensively studied as a model protein for LLPS. The protein contains a prion-like domain, two folded RNA binding domains, the RNA recognition motif (RRM) and a zinc finger domain (ZnF) and three arginine-glycine-glycine rich regions (RGG). The main molecular drivers and interactions underlying the LLPS phenomenon of FUS have been previously described, particularly for the LC domain. However, the role of the different regions in this structurally complex protein remains to be fully understood. In this work, the full-length protein was divided into two distinct previously unstudied constructs, namely RGG1-RRM-RGG2and RGG2-ZnF-RGG3. The LLPS propensity was examined, together with the molecular and structural drivers underlying the LLPS phenomenon of each construct. These objectives were addressed by performing turbidity assays and nuclear magnetic resonance spectroscopy (NMR). Based on the turbidity assays, the RGG1-RRM-RGG2construct appears to undergo LLPS when isolated. The folded RRM may play a direct role under certain conditions and RNA binding significantly affects the observed LLPS propensity. Moreover, at least ionic, and π-cation interactions act as drivers for the LLPS phenomenon of this domain. Through NMR spectroscopy, several residues of the folded RRM domain were significantly affected by temperature, sample cooling and reheating procedures and protein concentration. The specifically affected residues are likely to participate in protein-protein interactions, therefore establishing a hypothetical protein-protein interaction in the folded RRM domain. Due to the structural localization of the interface, the protein-protein interactions established by the folded RRM domain are not directly affected by RNA-binding. The RGG2-ZnF-RGG3 construct, as observed through NMR spectroscopy, presented an apparent cluster of H-bond performing residues within the folded ZnF domain, namely in the vicinity of the residues coordinating the Zn2+ ion. Furthermore, the construct did not appear to undergo LLPS under the tested conditions. To fully certify the absence of LLPS in this construct, further assays must be performed under distinct conditions. A previously unstudied FUS based construct revealed the capacity to undergo LLPS, driven at least by ionic, π-cation interactions and with direct participation of the folded RRM domain. The contribution of the RGG regions on the observed LLPS phenomenon remains to be elucidated. The tested RGG2-ZnF-RGG3did not undergo LLPS under the tested conditions. Further assays are necessary to evaluate the LLPS capacity of the construct

Metodologías innovadoras basadas en 19F-RMN de ligando y técnicas computacionales para el estudio de procesos de reconocimiento molecular azúcar-lectina

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 08-06-2021Carbohydrates play a central role in a large myriad of biological processes. They are found in all living organisms in nature, participating in different functions ranging from their use as energy source or as structural fragments, to infection-related processes in complex organisms. In vertebrates, they are located both in the cell surface and in the extracellular space, forming very diverse and intricate structures, but they are also present in the nucleus and cytoplasm of eukaryotic cells bound to proteins (glycoproteins). Their location almost ubiquitous in the organism confers them the capacity of mediate in a large number of ‘communication’ processes with other entities, for instance, in cell-cell, cell-molecule and cell-matrix interactions. In addition, carbohydrates intervene in molecular recognition processes between different organisms, such as the pathogen and parasite recognition by host cells...Los carbohidratos juegan un papel fundamental en una enorme variedad de procesos biológicos. Se encuentran en todos los organismos vivos en la naturaleza, donde intervienen en funciones que abarcan desde su uso como fuente de energía o como fragmentos estructurales, hasta procesos de infección en organismos superiores. En vertebrados, se localizan tanto en la superficie celular como en el espacio extracelular, formando estructuras muy diversas y complejas, pero también están presentes en el núcleo y citoplasma de células eucariotas unidos a proteínas (glicoproteínas). Su localización casi universal en el organismo les confiere la capacidad de intervenir en un gran número de procesos de ‘comunicación’ con otras entidades, por ejemplo, interacciones intercelulares, célula-molécula y célula-matriz extracelular. Además, los carbohidratos median procesos de reconocimiento molecular entre distintos organismos, como el reconocimiento de patógenos y parásitos por la célula de un huésped...Fac. de FarmaciaTRUEunpu

MOLECULAR DYNAMICS SIMULATIONS OF BIOLOGICAL MACROMOLECULES: APPLICATIONS TO STRUCTURAL VACCINOLOGY AND PEPTIDE DESIGN

This thesis work is splitted into two parts. The first one is about a computational method for epitope predictions on antigenic proteins, while the second one is related to the characterization of folding/unfolding processes of small natural polypeptides. Starting with the first topic, an increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. A public web server (BEPPE) has been implemented with MLCE method in order to make it available to the scientific community. Changing topic to folding/unfolding, the analysis of the folding mechanism in peptides adopting well defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of two 15-mer polypeptides (called QK and QK-L10A) homologue to the vascular endothelial growth factor binding region. In particular, on the basis of the temperature dependencies, we characterize the molecules through the combination of spectroscopic (CD and NMR) and computational analyses (MD) highlighting their folding/unfolding steps and how these structures can be used in peptide design

Security and Privacy in Molecular Communication and Networking: Opportunities and Challenges

International audienceMolecular Communication (MC) is an emerging andpromising communication paradigm for several multi-disciplinarydomains like bio-medical, industry and military. Differently to thetraditional communication paradigm, the information is encodedon the molecules, that are then used as carriers of information.Novel approaches related to this new communication paradigmhave been proposed, mainly focusing on architectural aspects andcategorization of potential applications. So far, security and privacyaspects related to the molecular communication systems havenot been investigated at all and represent an open question thatneed to be addressed. The main motivation of this paper lies onproviding some first insights about security and privacy aspects ofMC systems, by highlighting the open issues and challenges andabove all by outlining some specific directions of potential solutions.Existing cryptographicmethods and security approaches arenot suitable for MC systems since do not consider the pecific issuesand challenges, that need ad-hoc solutions.We will discuss directionsin terms of potential solutions by trying to highlight themain advantages and potential drawbacks for each direction considered.We will try to answer to the main questions: 1) why thissolution can be exploited in the MC field to safeguard the systemand its reliability? 2) which are the main issues related to the specificapproach

Los receptores para el reconocimiento de patrones moleculares: aportaciones de la química computacional para el diseño de fármacos y la modulación de la inmunidad innata

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 18/11/2019In this Thesis we have aimed the study of the molecular recognition processes of receptors involved in the innate immunity. More concretely, we have focused in two different types of lectins, Galectins and DC-SIGN, and in Toll-like receptor 4. We have made use of computational techniques, including docking and virtual screening, molecular dynamics simulations, conformational analysis and quantum mechanics calculations. The work has been organized into several chapters that are summarized as follows: Chapter 1 corresponds to the current knowledge and perspectives about receptors related to immunity, in particular: galectins, DC-SIGN, and Toll-like receptor 4, corresponding to the molecular recognition events and modulation by small molecules. Chapter 2 describes the state-of-the-art methods in molecular modeling and computational chemistry applied to the study of molecular recognition processes and drug design...En esta tesis hemos estudiado los procesos reconocimiento molecular de receptores involucrados en la inmunidad innata. Más concretamente, nos hemos centrado en dos tipos diferentes de lectinas, Galectinas y DC-SIGN, y en el receptor Toll-like 4 (TLR4). Hemos utilizado técnicas computacionales, incluyendo docking y cribado virtual, simulaciones de dinámica molecular, análisis conformacional y cálculos de mecánica cuántica. El trabajo se ha organizado en diferentes capítulos que se resumen como sigue: El capítulo 1 corresponde al estado del arte y las perspectivas relacionadas con los estudios de reconocimiento molecular proteína-carbohidrato y diseño de nuevos moduladores con actividad biológica en receptores de la inmunidad, en particular galectinas, DC-SIGN y el receptor Toll-like 4. El capítulo 2 describe el estado actual de los métodos en modelado molecular y química computacional aplicados al estudio de los procesos de reconocimiento molecular y diseño de fármacos...Fac. de FarmaciaTRUEunpu

Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.The Icelandic Research Fund [grant number 162977-051

Computational Studies on the Relation Between Macromolecular Dynamics and Protein Binding and Function

Computational methods can help to better understand and analyze the interaction of proteins and their binding partners. This interaction is influenced by many factors, including specific sequence variants, the dynamics and electrostatics of the proteins, as well as further physicochemical properties of the corresponding binding partners. A detailed investigation of these different, and often complicated, properties helps to better understand the functionality of proteins, for which the interaction with other molecules plays a crucial role. The work presented here provides new methodologies, implemented in webservers and software, which assist during the analysis of proteins. Furthermore, in an application case, computational methods and analyses in combination with experimental results were used to detect a specific interaction network of proteins. The new ProSAT+ webserver enables the visualization of protein sequence annotations in the context of the three–dimensional protein structure and contains additional options for visualizing and sharing protein annotations. The sequence information allows an easy, but extensive analysis of proteins. The functionality of the ProSAT+ webserver can be integrated into other webservers, which was done in the case of the two other webservers for the analysis of protein binding pockets described here. A tool for the LigDig webserver was developed that provides the comparison of protein binding pockets by the alignment and visualization of the binding pockets based on an existing algorithm. The new TRAPP webserver assists in the analysis of protein binding pocket dynamics. The existing TRAPP software was used, and a user web interface was implemented to simplify the usability. Additional new functionalities were also developed, such as the visualization of protein sequence conservation in context of all other TRAPP results in the three–dimensional structure. This allows the detection of conserved or non–conserved regions inside the binding pocket, which might influence the dynamics of the pocket. This newly gained information can be used during the process of designing selective inhibitors. During the protein disaggregation process, members from different classes of the so-called J–protein (HSP40) co–chaperones play a crucial role. The synergetic application of different computational methods and experiments enabled the detection of an interclass specific J–protein interaction and indicated that the interaction evolved to enable a high efficiency in the disaggregation process. The resulting data of performed protein domain docking simulations required an update of the standard clustering workflow. This new methodology can be applied for protein docking in cases that have problems with multiple, weakly specific interaction sites. The work presented here facilitates in many ways the analysis of proteins, including their structure and sequence features, as well as, their dynamics and interactions with their binding partners. The new methods are provided as webservers and therefore are accessible, and easy to use for all researchers. This can assist in many research projects and provide relevant information. The analyses of the J–proteins improved the knowledge about their biological role and functionality, and therefore provide an important contribution for a better understanding of the overall protein disaggregation process