Evaluation of the physical properties of experimental macromolecular crowding systems

Thrithamara Ranganathan, Venketesh (2022) Evaluation of the physical properties of experimental macromolecular crowding systems. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Biological cells are crowded environments consisting of both large and small molecules. The macromolecular crowding observed in biological cells is likely very important to the structure and function of a living cell. Since the macromolecules can interact with each other, this makes the system complex and there remain several open questions. In the laboratory, artificial crowder molecules can be used to create experimental model systems that mimic the cellular environment. Artificial crowders such as the polysaccharide, Ficoll, have long been assumed to be compact and colloidal, but a holistic understanding of its structure and dynamics is lacking. This thesis investigates the structure of Ficoll using multiple experimental techniques. We report rheology, small angle neutron scattering, self diffusion and relaxation measurements using nuclear magnetic resonance experiments on two widely used artificial crowder molecules, Ficoll-70 and Ficoll-400. Our results, combining measures of structure, diffusion, relaxation and rheology, show that Ficolls are more polymer like than colloid like. Importantly, we find that the self-diffusion of HDO molecules in the suspension is an efficient probe to evaluate volume occupancy of the suspension under investigation. We then evaluate the physical properties of a protein crowder solution, BSA, and phytoglycogen, a natural plant based dendrimer using the methods developed to evaluate the Ficoll suspension properties. For all crowders, we find that the self diffusion coefficient decreases exponentially with a characteristic concentration of 10-12 wt %. We also observe that the NMR transverse relaxation of the solvent is a sensitive, independent measure of water confinement, which can be correlated with suspension rheology and self diffusion. To summarize, the highlight of this thesis is that structural and dynamical methods that report on macromolecules as well as solvent can provide a more complete view of macromolecular crowding.

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