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Transcription factors regulate gene activity by binding specific regions of genomic DNA thanks to a subtle interplay of specific and nonspecific interactions that is challenging to quantify. Here, we exploit Reflective Phantom Interface (RPI), a label-free biosensor based on optical reflectivity, to investigate the binding of the N-terminal domain of Gal4, a well-known gene regulator, to double-stranded DNA fragments containing or not its consensus sequence. The analysis of RPI-binding curves provides interaction strength and kinetics and their dependence on temperature and ionic strength. We found that the binding of Gal4 to its cognate site is stronger, as expected, but also markedly slower. We performed a combined analysis of specific and nonspecific binding—equilibrium and kinetics—by means of a simple model based on nested potential wells and found that the free energy gap between specific and nonspecific binding is of the order of one kcal/mol only. We investigated the origin of such a small value by performing all-atom molecular dynamics simulations of Gal4–DNA interactions. We found a strong enthalpy–entropy compensation, by which the binding of Gal4 to its cognate sequence entails a DNA bending and a striking conformational freezing, which could be instrumental in the biological function of Gal4.

Adenylyl cyclases (ACs) catalyze the biosynthesis of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and play an important role in many signal transduction pathways. The enzymatic activity of ACs is carefully controlled by a variety of molecules, including G-protein subunits that can both stimulate and inhibit cAMP production. Using homology models developed from existing structural data, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase and on its complexes with ATP and with the stimulatory G-protein subunit Gsα. The results show that both ATP and Gsα binding have significant effects on the structure and flexibility of adenylyl cyclase. New data on ATP bound to AC5 in the absence of Gsα notably help to explain how Gsα binding enhances enzyme activity and could aid product release. Simulations also suggest a possible coupling between ATP binding and interactions with the inhibitory G-protein subunit Gαi.

Hierarchical self-assembly is a process in which molecular building blocks form intermediate structures that self organise at macroscopic level. Remarkable examples can be found in nature, like, for instance, DNAs or viruses. Self-assembly offers interesting strategies to build new complex materials: Therefore, it is very important to understand its mechanism to design and control molecular architectures and to build structures with desired properties and morphologies. A major question is how the shape of the building blocks influences self- assembly. In this context, chirality plays a crucial role: It is extremely sensitive to subtleties on the molecular scale and can guide self-assembly; furthermore, chirality can act as an amplifier of changes that occur at the molecular level. From the theoretical point of view, the difficulty derives from the need of multiscale methods and models, able to connect the different length scales. To take into account the relationship among the building blocks, their supramolecular organization, and the properties of the aggregates, a detailed representation of intermolecular interactions is needed: This description has to be integrated into a suitable modeling of the system behaviour on a much longer length scale. This thesis deals with the development and implementation of models for chirality propagation from the molecular to the meso- and macroscopic levels in self-assembling systems. In particular, the research has been carried out along three lines. The first deals with self-assembly of hard helices, leading to the formation of anisotropic phases of vari- ous symmetry. The second topic is the linear aggregation and formation of liquid crystal phases by double-stranded nucleic acid oligomers (dsNA): The relationship between the sequence of oligonucleotides, their self-assembly and the properties of their cholesteric phase is investigated. The last topic is the aggregation of porphyrin-polypeptide conjugates in water. Depending on the problem and the length scale, we used different theoretical and com- putational methods, in particular: Statistical theories of liquids and molecular dynamics simulations (both atomistic and coarse-grained). The first and third topics have been carried out in collaboration with experimentalists, while for the second other groups of theoreticians have been involved. This thesis is organized in three parts. In Chapter 1, the concepts of self-assembling, chi- rality propagation and multiscale modeling are introduced. Moreover this Chapter presents an outline of the main properties of liquid crystals. The first Part, from Chapter 2 to Chapter 4, presents the study on the anisotropic phases formed by hard helices. Chapter 2 presents a study of the nematic phase, using an Onsager- like theory. The theoretical results are compared with Monte Carlo simulations. In Chapter 3, a theoretical model for the cholesteric phase is presented and used to investigate the relationship between the helical shape and the properties of the cholesteric phase. In Chapter 4, the complete phase diagram of hard helices is presented, together with the characterization of a novel chiral nematic phase. The second Part deals with liquid crystal phases formed by dsNA. Chapter 5 focuses on the relation between the sequence of oligonucleotides and their organization in the cholesteric phase, using a molecular theory and coarse-grained modelling based on sequence dependent structural data. Chapter 6 describes the theoretical model for the cholesteric phase formed by self-assembling oligomers, which integrates the theory for cholesteric order presented in Chapter 3 with that for linear aggregation in the nematic phase. The last Part, from Chapter 7 to Chapter 9, deals with the aggregation of porphyrin- peptide conjugates in water. In Chapter 7 the main concepts of circular dichroism are introduced and the state-of-the-art of self-assembly of porphyrins is reviewed. Chapter 8 describes atomistic molecular dynamics simulation of aggregates of porphyrin-peptide conjugates. Chapter 9 presents a study of the same systems by coarse-grained molecular dynamics simulations, using the MARTINI model. Finally, Chapter 10 presents a summary, which highlights the relevant results obtained in this Thesis, and three Appendices follow.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p &lt, 0.05) and an increase in pseudo-tube width (41 ± 4.5%, p &lt, 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p &lt, 0.01), which is associated to a higher Ca2+ mobilization (p &lt, 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p &lt, 0.001, p &lt, 0.01, and p &lt, 0.01, respectively) as well as actin/tubulin distribution (p &lt, 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p &lt, 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.