Dynamic force spectroscopy and folding kinetics in molecular systems

Dynamic force spectroscopy and folding kinetics in molecular systems Anna Alemany Codification of genetic information; regulation of gene expression; transport of nutrients inside cells; immune protection against infectious agents; transduction of external signals. These are some of the crucial processes that take place in living organisms at the molecular level. A deep understanding of how these phenomena occur is vital to get a precise knowledge of the laws governing the microscopic world and understand, prevent and even find cures for many illnesses with an origin in the molecular scale. Single-molecule experiments emerge as a powerful and versatile tool to investigate molecular processes at the level of individual molecules and trajectories with unprecedented spatial and temporal resolution. A paradigmatic experimental technique is given by ? optical tweezers? (OT), which consist of a laser beam that captures micron-sized plastic beads using light momentum conservation. This instrument makes it possible to manipulate with nanometric precision a biomolecule and exert forces on it in the range of [0-100] pN. The diversity of systems being studied using optical tweezers increases every day. In this thesis, OT are used to unravel the mechanisms of unfolding and folding of several small nucleic acid hairpins and a protein when a force is applied to their ends. Moreover, single antigen-antibody bonds are investigated by qualitatively measuring the correlation between bond affinity and bond elasticity. All single-molecule experiments have in common the relevant role played by thermal fluctuations. These are crucial at the microscopic scale and contain overriding thermodynamic and kinetic information of molecular systems. The study of the characteristic forces at which molecular cooperative transitions occur, such as molecular unfolding or bond dissociation, is known as dynamic force spectroscopy (DFS). In this thesis DFS combined with Markov models are widely used to characterize the unfolding/folding reaction pathway, the transition states present in the molecular free-energy landscape, and the elastic, kinetic and thermodynamic properties of a protein, the antigen-antibody bond and nucleic acid hairpins under different conditions. A general result is that non-equilibrium DFS methods provide an excellent platform to extract thermodynamic properties of molecular states that can only be observed under dynamical conditions (that is, they are never observed in equilibrium at reasonable time scales), such as intermediate or bound configurations. An alternative method to extract thermodynamic properties of molecular systems is the use of fluctuation relations (FR). These are mathematical identities that relate non-equilibrium work measurements to free-energy differences. When an irreversible transformation is mechanically induced in an otherwise full-equilibrated molecular system, a work is performed between an initial and a final state that differs in each independent repetition of the experiment. FR relate a collection of these work measurements to the difference in free energy between the initial and the final states, independently of the molecular reaction pathway. If the system is initially found at a partially equilibrated state, an extended version of FR can be used to measure its free energy of formation. This grants access to the thermodynamic properties of misfolded and intermediate states that are rarely sampled in full equilibrium. Additionally, the extended FR allow us to reconstruct the free-energy branches of molecular states observed during non-equilibrium experiments from irreversible work measurements. Hence, relative stabilities between molecular native, unfolded, misfolded an intermediate states can be compared at different stages of the irreversible experiment. Results in this thesis pave the way to characterize the thermodynamics and kinetics of complex molecular process that occur under partial equilibrium conditions (as is the case in living organisms), using both DFS and FR. Examples are found in many kinetic states related to intermolecular binding or in transient non-equilibrium states occurring in polymerization reactions (e.g. translocating). Hence the influence of the findings and methods developed in this thesis remains to be seen.