Fluctuations in biological molecules: tools to probe mechanical and structural properties of DNA and proteins

The traditional boundary between hard sciences (physics and mathematics) and soft sciences (chemistry and biology) is progressively fading away as the complexity inherent in the biological world is understood and mapped out thanks to a joint attack on two fronts. On the one side more quantitative experiments allow to investigate the details of the atomic structures of biological molecules and to measure with greater precision the laws of interaction among dierent molecules; on the other side, the massive introduction of information technology in the management and catalogation of the multitude of molecular components found inside a cell is allowing to gain deep insights in the complex dynamic equilibrium that regulates the network of interactions amog different molecules. The work described in this thesis concerns the first side of the battlefield: the development of new techniques to allow quantitative measurements of biologically relevant quantities. The work consisted in the design, construction and validation of three different experiments dealing with proteins and DNA mechanics. Key components of the cellular microcosmos, DNA and proteins are large macromolecules that constantly interact and accomplish most of the tasks needed by the cell to survive. The first part of the thesis summarises the known properties of these molecules and introduces the motivations driving the designed experiments. Proteins catalyse chemical reactions in the cell and their threedimensional conguration gives each of them its specic function. The connections between structural and chemical properties of a protein are a subject largely unexplored.The second part of the thesis describes an experiment based on single molecule fluorescence microscopy designed to explore the dynamics of fluctuations of catalytic activity of a single enzyme. The experiments described in this part have not yet given the hoped results. However part of the preliminary considerations done when building these setup were used to write the article F. Mosconi et al. "Some nonlinear challenges in biology", Nonlinearity 21 (2008) T131-T147. DNA stores the genetic information needed by the cell to accomplish its tasks, and such information must be physically stored, read, written and restored in different times during the cell cycle. The importance of a proper knowledge of its mechanical properties is fundamental if its interaction with proteins is to be understood. The third part of this thesis describes two different experiments based on the magnetic tweezers micro-manipulation technique, that attempt to measure some not yet entirely characterised mechanical properties of DNA. The two experiments presented in this part gave interesting results. A new determination of the biologically relevant parameter C, the twist modulus of DNA was obtained developing a novel type of analysis of data collected using the standard magnetic tweezers apparatus. Also, a new type of "soft" magnetic tweezers that allows the simultaneous application of an external force and an external torque has been developed and validated to measure the torque response of a DNA molecule. The results described in this part of the thesis are summarised in two papers that are ready to be submitted.