Structural biology provides fundamental insight into protein function. One underlying mechanistic principle of protein function may be protonation dynamics, that is structural alterations related to changes in protonation states. The interplay between proton exchange processes and biomacromolecular structure, i.e. function, needs to be further established in life sciences. Protonation dynamics can be studied by different theoretical, biophysical and biochemical approaches. Nuclear magnetic resonance (NMR) spectroscopy is a powerful method to detect protons directly and to elucidate structural features related to changes in protonation states. With this work, two advanced solid-state magic angle spinning (MAS) NMR spectroscopy approaches, proton detection and dynamic nuclear polarization (DNP), are applied to study protonation dynamics in large proteins and membrane proteins. Proton detection combined with fast MAS is employed to obtain spatial information on hydrogen atoms. A case study is presented, proposing a proton-detected solid-state MAS NMR experiment that enables the observation of hydrogen bonds in a model protein, a microcrystalline preparation of the chicken a-spectrin SH3 domain. It is used to elucidate hydrogen bond patterns in secondary structure elements; in a modified version, it may be applied to detect such patterns in amino acid side chains. In a pilot study, chemical exchange of protons is investigated with the light-driven proton pump bacteriorhodopsin as an example. Proton relocation was observed inside the channel at three functionally-relevant key amino acid side chains, which are involved in the proton transport pathway of bacteriorhodopsin. We detect a protonated form of R82 suggesting its involvement in the proton pumping process, and we notice proton delocalization between the carboxylic moieties of both D85 and D96 and water. Furthermore, a study on the soluble extra-cellular domain of the neonatal Fc receptor is presented as a methodologically oriented application of fast MAS NMR. This receptor interacts with Immunoglobulin G in a pH-dependent manner and thus via changes in protonatable amino acid side chains. Hence, protonation dynamics is fundamentally involved in this protein-protein interaction. The study aims to develop a small molecule and characterize its binding structurally to explore possibilities for inhibition of the interaction with Immunoglobulin G. The application of NMR at 100 kHz MAS to the sedimented, fully protonated receptor was crucial to obtain chemical shift perturbations upon binding of the identified ligand as a prerequisite for optimization of the compound. This study introduces an innovative approach to investigate soluble proteins expressed in mammalian cells by proton-detected MAS NMR without the need for deuteration, making a variety of protein classes accessible to NMR studies. As demonstrated in this thesis, structural features related to protonation dynamics can be difficult to observe by conventional NMR techniques due to low sensitivity. Furthermore, halting exchange processes by freezing samples may facilitate protonation dynamics investigations. The DNP technology is therefore further developed in two studies using proline standard samples and the SH3 domain. We present a high-temperature approach employing deuterated biradicals and introduce a novel highly water-soluble biradical, called bcTol. The use of deuterated TOTAPOL isotopologues resulted in a 15-fold increase in sensitivity at 200 K, thereby facilitating the acquisition of multidimensional spectra with improved resolution at this temperature. The new biradical bcTol is demonstrated to be a promising and efficient polarizing agent in biomolecular investigations, easy-to-handle and showing an improved performance compared to other known biradicals. These methodological achievements enabled studies of protonation dynamics in complex protein systems. In this context, structure-related features in the chromophores of channelrhodopsin and the phytochrome photoreceptor Cph1 are investigated via the utilization of DNP. The enhancement in sensitivity helped to elucidate the retinal configuration to be all-trans in dark-adapted channelrhodopsin, which is an important feature of the photocycle. It allowed us to discuss the chromophore structural changes enabling proton conductance across the membrane. In Cph1, the application of DNP was critical to obtain chemical shift assignments of the phycocyanobilin chromophore nitrogens. This provided insight into the water molecule distribution in the chromophore binding pocket and the localization of the positive charge in phycocyanobilin. These findings help to understand the chromophore changes and possible proton exchange pathways during molecular action of phytochrome photoreceptors.
