Solid-state NMR studies on therapeutic peptides

Abstract

Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy has emerged as a powerful tool that allows the study of proteins and protein complexes in their native environment. Such information can be of great value especially in the field of drug development. Moreover, ssNMR enables the study of protein dynamics, drug – target interactions and heterogeneous systems. In this thesis, we have investigated the plectasin – lipid II complex, peptidic hydrogels for spinal cord tissue engineering, and modal gating behaviour in the K+ channel KcsA using state-of-the-art ssNMR experiments. Antimicrobial resistance is a growing threat to human health worldwide which urgently calls for novel antibiotics that act through unexploited pathways. A validated and promising target for antibiotic design is lipid II, an essential precursor in the biosynthesis of the peptidoglycan network. However, structural data on the drug – lipid II complex are scarce and unavailable in its physiologically relevant membrane environment. In Chapters 2 and 3, we exploit the plectasin – lipid II complex in membranes. We analysed the structure and dynamics of plectasin in the unbound and bound state in great detail using high resolution ssNMR. Comparing plectasin in the two states revealed strong structural changes in previously ignored structural regions of plectasin such as the αβ-loop. Moreover, using 15N,13C- labelled lipid II enabled us to experimentally determine the complex interface. Overall, we have found stark differences between the mode of action of plectasin in micelles and in membranes, which further underlines the need of physiologically relevant studies for drug design purposes. Material properties of tissue engineering scaffolds dominate the behaviour of stem cells and are inherent to the success of tissue engineering. In Chapter 4 we explore the feasibility of studying the mechanical properties of a series of peptidic hydrogels, designed for neural tissue regeneration for treatment of spinal cord injuries, using modern ssNMR at natural abundance. We could not only describe material properties such as the rigidity and assembly at high resolution, but we could also identify the scaffold conformational heterogeneity as novel and decisive design parameter for the functionality of scaffolds. Overall, we have demonstrated that ssNMR methods pave the way to obtain atomic level insights into mechanical properties of tissue engineering scaffolds. Modal gating shifts are a widespread regulatory phenomenon in many ion channels. However, the molecular underpinning of these shifts remains unknown. In Chapter 5 we explore the molecular origin of modal gating behaviour in the archetypical K+ channel KcsA in native-like membranes using extensive ssNMR dynamics studies. We characterized the conformation and dynamics of mutant channels that represent the different modal gating modes. By integrating our ssNMR data with MD simulations, we discovered that shifts in the selectivity filter dynamics, caused by fluctuations in the water and hydrogen bonding network, are the driving forces for modal gating behaviour in KcsA. Thereby, our results provide a foundation for an improved understanding of K+ channels in general.