Investigation of cell interfaces and strained tissues with solid-state NMR

Abstract

Solid-state nuclear magnetic resonance (ssNMR) is an immensely powerful tool for structural biology, which can provide insights into the composition, structure, dynamics of biological components. In combination with 13C amino acid enrichment of cell fibroblasts in vitro, ssNMR has been successfully used to study insoluble biological components such as the extracellular matrix (ECM). This thesis, aims to develop innovative NMR methods to expand the range of what is currently achievable with NMR. First, we introduce NMR concepts that will be used in the remaining part of the work, with a particular focus on the characterisation of spin diffusion, a means to transport magnetisation that is used extensively in later chapters. The following part aims to achieve spatial selectivity on cell samples with ssNMR. The ECM surrounding cells provides an ideal environment that fosters cell function. The ECM is often studied after extensive and denaturing isolation and little is known about how cells interact with the ECM at the molecular level for a lack of appropriate methods to study it. In the first part of this thesis are developed solid-state NMR methods that can be used to achieve spatial selectivity with ssNMR, without the need for an extensive – and potentially damaging – sample preparation that isolates the region of interest. First, Goldman-Shen-like experiments that utilise 1H spin diffusion to transport NMR signal from cell membranes to nearby ECM components, are being used to record interface-edited ssNMR spectra. Second, the NMR signal enhancement obtained from dynamic nuclear polarisation (DNP) using a polarising agent that colocalises close to cell membranes is used to record ssNMR spectra selective to cell plasma membranes. Third, a technique aiming to suppress intracellular NMR signals is being developed using internalised gadolinium relaxation agents. In the last part, we develop NMR methods to study tissues under strain. Tendons are primarily made of collagen protein, forming a structure that is remarkably resilient to mechanical damage despite the constant stress that is being exerted on it in our everyday lives. Innovative ssNMR-based methods would be useful to study and rationalise the effect of mechanical strain on tendon collagen. For that purpose, tendons are studied in the first place under strain, primarily using ssNMR, microscopy and chemical shift calculations. In the following chapter, mechanochemical products and structural changes that occur when tendons are brought beyond their breaking point are being studied with ssNMR.