Understanding the structure and dynamics of proteins is essential to understanding their roles and functions in these physiological processes. In this thesis, I describe the implementation of an ultrafast nonlinear spectroscopic technique, two-dimensional infrared (2D IR) spectroscopy to probe the structure and dynamics of ion channels and amyloid fibers. Regarding ion channels, I describe the combination of semisynthesis, 2D IR spectroscopy and molecular dynamic (MD) simulations in addressing the longstanding question of ion permeation through the selectivity filter of a potassium ion channel. I show that ions and water alternate through the filter and that these ions cannot occupy adjacent binding sites. Furthermore, 2D IR experiments revealed a flipped state that is predicted by MD simulations but not observed in x-ray crystallography. In another aspect of this work, we show that the collapsed state of the filter is structurally different in low K+ and low pH. Moreover, our work also reveals how the large conformational motions of the protein are coupled to structural changes in the selectivity filter, as evidenced by a change in the ion occupancy. In a second research direction, I developed an optical technique to quantify photoactivatable fluorophores with fluorescence microscopy. This technique allows for the quantification of a limitless number of fluorophores, and corrects for stochastic events such as fluorescence intermittency. This work can be extended to the study of amyloids, where determining the number of proteins in a prefibrillar aggregates is necessary for understanding their roles in amyloid related diseases. Finally, using 2D IR spectroscopy we describe the effect of common solvents on the anharmonicity of small molecule chromophores. The data indicates that the carbonyl anharmonicity, and, subsequently, the Stark tuning rate, is an intrinsic property of the carbonyl vibrational probes, which have important implications on the interpretation of carbonyl vibrational frequency shifts in the condensed phase.

One key property of metal-organic frameworks (MOFs) are their structural elasticity. IHere we show that 2D IR spectroscopy with pulse-shaping techniques can probe the ultrafast structural fluctuations of MOFs. 2D IR data, obtained from a vibrational probe attached to the linkers of UiO-66 MOF in low concentration, revealed that the structural fluctuations have time constants of 7 and 670 ps with no solvent. Filling the MOF pores with dimethylformamide (DMF) slows the structural fluctuations by reducing the ability of the MOF to undergo deformations, and the dynamics of the DMF molecules are also greatly restricted. Finally, methodology advances were required to remove the severe light scattering caused by the macroscopic-sized MOF particles, eliminate interfering oscillatory components from the 2D IR data, and address Förster vibrational excitation transfer.

The advent of laser-based sources of ultrafast infrared pulses has extended the study of very fast molecular dynamics to the observation of processes manifested through their effects on the vibrations of molecules. In addition, non-linear infrared spectroscopic techniques make it possible to examine intra- and intermolecular interactions and how su

This thesis presents a highly innovative study of the ultrafast structural and vibrational dynamics of hydrated phospholipids, the basic constituents of cell membranes. As a novel approach to the water-phospholipid interface, the author studies phosphate vibrations using the most advanced methods of nonlinear vibrational spectroscopy, including femtosecond two-dimensional infrared spectroscopy. He shows for the first time that the structure of interfacial water undergoes very limited fluctuations on a 300 fs time scale and that the lifetimes of hydrogen bonds with the phospholipid are typically longer than 10 ps. Such properties originate from the steric hindrance of water fluctuations at the interface and the orienting action of strong electric fields from the phospholipid head group dipoles. In an extensive series of additional experiments, the vibrational lifetimes of the different vibrations and the processes of energy dissipation are elucidated in detail.

Understanding most of the physical and chemical phenomena determining the world around us requires the possibility to interrogate their main characters on their natural scale in space and time. The insulating or conductive behavior of matter, its magnetic properties or the nature of chemical bonds are strongly dependent on the nuclear and electronic structure of the atoms, molecules or solids considered. Hence, tools are needed to probe electrons and nuclei directly at the atomic scale with a temporal resolution allowing the observation of electron dynamics (on the attosecond-to-femtosecond timescale) and structural dynamics (on the femtosecond-to-picosecond timescale) in real time.Attosecond science offers unique opportunities to investigate electronic and structural dynamics at the heart of important processes in atomic, molecular and solid-state physics. The generation of attosecond bursts of light, in the form of train of pulses or of isolated pulses, has been achieved on table-top sources by exploiting the high-order harmonic generation (HHG) process. The photons constituting the attosecond emission have energies that range from the extreme ultra-violet (XUV) up to the soft X-ray (SXR) region of the spectrum, allowing to interrogate the electronic structure of the probed material directly at the level of the inner electronic shells. Because of this property of accessing the characteristic electronic structure of the elements constituting the target, XUV and, especially, SXR spectroscopy are considered element-specific techniques. Attosecond pulses have already proven to be able to observe ultrafast phenomena in atoms, molecules or solids previously inaccessible.In this thesis, the application of time-resolved X-ray absorption fine-structure (XAFS) spectroscopy using attosecond SXR pulses to the study of carrier and structural dynamics in graphite is reported. In chapter 1, an introduction to the field of attoscience and the presentation of the state of the art of ultrafast dynamics in graphite are given. The established technique to generate attosecond pulses is described and a review of the most significant application of attosecond pulses to the study of electron dynamics is presented. The electronic and structural properties of graphite are then discussed, highlighting some of the most representative experiments detecting electron and lattice dynamics.The experimental setup developed at ICFO in the group of Prof. Dr. Jens Biegert and used for this Ph.D. thesis project is described in details in chapter 2. The system needed for the generation, propagation and detection of the attosecond SXR radiation is presented. The performances of the SXR source in terms of spectral tunability, photon flux and stability are discussed. The implementation of a IR pump - SXR probe scheme is reported, allowing beams' recombination in both collinear and non-collinear fashion. To conclude, the results of an attosecond streaking experiment are presented, through which a temporal characterization of the HHG emission has been achieved.A discussion on the spectroscopic capabilities of XAFS technique to interrogate the electronic and lattice structure of the observed material is presented in chapter 3. The potential of this technique has been demonstrated with an experimental investigation of a graphite thin film, with the results showing the possibility to probe the first unoccupied electronic bands and the characteristic distances defining the lattice structure.Finally, the XAFS capabilities have been exploited in a time-resolved experimental study of graphite to observe light-induced carrier and lattice dynamics, presented in chapter 4. The interpretation of the experimental data reveals insights on the ultrafast interaction of the pump laser field with charge carriers and on the effects of carrier-carrier and carrier-phonon scattering following photoexcitation.