3 research outputs found

    Predicting structural determinants and Ligand poses in proteins involved in neurological diseases: bioinformatics and molecular simulation studies

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    Part I presents the computational tools used in this work: the comparative modeling and molecular docking approaches along with molecular dynamics. Part II presents structural predictions of Ca2+-binding domains in Ca2+-gated channels. A detailed description of the structure and function of these proteins can be found in the following Chapters. Chapter 4 focuses on human large conductance Ca2+- and voltage-gated potassium channel (hBKCa). Bioinformatics approaches and MD simulations were used to construct models of two domains important for Ca2+ binding and channel gating, namely the Regulator of Conductance for K+ (RCK1) and the so called calcium bowl. The relevance of these models for interpreting the available molecular biology data is then discussed. Chapter 5 deals with bestrophins, a recently discovered family of Cl 12 channels. Bestrophins feature a well conserved Asp-rich tract in their C-terminal part, which is homologous to Ca2+-binding motifs in calcium bowl of hBKCa. Based on these considerations, we constructed homology models of human bestrophin-1 Asp-rich domain. MD simulations and free energy calculations were used to identify Asp and Glu residues binding Ca2+ and to predict eects of their mutations to Ala. My work, performed in collaboration with C. Anselmi (SISSA/ISAS), was complemented by free energy calculations carried out by F. Pietrucci (SISSA/ISAS). Selected mutations were investigated by electrophysiological experiments performed by Prof. A. Menini, J. Rievaj, F. W. Grillo, and A. Boccaccio (SISSA/ISAS). The model of Asp-rich domain was then validated against experimental results. Part III is devoted to the prion protein. In this Part, Chapter 6 presents in vitro studies of D18scFv anti-prion effects performed by groups of Prof. C. Zurzolo (Institut Pasteur, Paris, France), Prof. G. Legname (SISSA/ISAS), L. Zentilin and M. Giacca (ICGEB, Trieste, Italy) and by Prof. S. B. Prusiner (Institute for Neurodegenerative Diseases, University of California San Francisco, U.S.A.) and structural prediction of a complex between the small antibody fragment (D18scFv) and PrPC. The complex was modeled using bioinformatics approaches. Initially, the D18scFv fragment alone was modeled based on a similar antibody-fragment template and then docked with prion protein. Based on this, interactions relevant for the recognition between the two proteins and for the mechanism of action of D18scFv are discussed. Chapter 7 describes a computational protocol for the design of ligands targeting cavity-less proteins, like most proteins involved in neurodegenerative diseases. Molecular docking methods are combined with MD simulations and free energy calculations using the metadynamics method [33, 34] to gain insights in ligand binding to such proteins, in our case to prion protein. We focused on a compound showing antiprion activity in vitro. Ligand-target interactions and ligand binding affinity as emerged by using our approach are compared with the available NMR data [35] and experimental constant of dissociation [35]. In this work, also other two students and one postdoc were involved beside myself, namely S. Bongarzone, G. Rossetti and X. Biarnes (SISSA/ISAS). Finally, the conclusions are drawn in the last Chapter. The thesis closes with the List of publications and with the Acknowledgments

    Piezoelectric microcantilever serum protein detector

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    The development of a serum protein detector will provide opportunities for better screening of at‐risk cancer patients, tighter surveillance of disease recurrence and better monitoring of treatment. An integrated system that can process clinical samples for a number of different types of biomarkers would be a useful tool in the early detection of cancer. Also, screening iomarkers such as antibodies in serum would provide clinicians with information regarding the patient’s response to treatment. Therefore, the goal of this study is to develop a sensor which can be used for rapid, all‐electrical, real‐time, label‐fee, in‐situ, specific quantification of cancer markers, e.g., human epidermal receptor 2 (Her2)or antibodies, in serum. To achieve this end, piezoelectric microcantilever sensors (PEMS) were constructed using an 8 ÎŒm thick lead magnesium niobate‐lead titanate (PMN‐PT) freestanding film as the piezoelectric layer. The desired limit of detection is on the order of pg/mL. In order to achieve this goal the higher frequency lateral extension modes were used. Also, as the driving and sensing of the PEMS is electrical, the PEMS must be insulated in a manner that allows it to function in aqueous solutions. The insulation layer must also be compatible with standardized bioconjugation techniques. Finally, detection of both cancer antigens and antibodies in serum was carried out, and the results were compared to a standard commercialized protocol. PEMS have demonstrated the capability of detecting Her2 at a concentration of 5 pg/mL in diluted human serum (1:40) in less than 1 hour. The approach can be easily translated into the clinical setting because the sensitivity is more than sufficient for monitoring prognosis of breast cancer patients. In addition to Her2 detection, antibodies in serum were assayed in order to demonstrate the feasibility of monitoring the immune response for antibody‐dependent cellular cytotoxicity (ADCC) in patients on antibody therapies such as Herceptin and Cetuximab. The PEMS displayed a limit of detection of 100 fg/mL, which was 100 times lower than the current methods of protein detection in serum, such as ELISA. Furthermore, the sensitivity of the PEMS device allows it to be capable of determining the dissociation constant, Kd, of selective receptors such as antibodies. Using the dose response trials of Her2, Kd has been deduced for H3 scFv, and Herceptin, a commercial antibody specific for Her2.Ph.D., Materials Engineering -- Drexel University, 200
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