3 research outputs found
Predicting structural determinants and Ligand poses in proteins involved in neurological diseases: bioinformatics and molecular simulation studies
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
The development of a serum protein detector will provide opportunities for better screening of atârisk 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 patientâs response to treatment. Therefore, the goal of this study is to develop a sensor which can be used for rapid, allâelectrical, realâtime, labelâfee, inâsitu, 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 niobateâlead titanate (PMNâPT) 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 antibodyâdependent 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