Date of Award

12-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering and Sciences

First Advisor

Boris Akhremitchev

Second Advisor

Nasri Nesnas

Third Advisor

Yi Liao

Fourth Advisor

David Carroll

Abstract

Over the last couple of decades, there has been rapidly growing interest in research of natively unfolded proteins. Some proteins from this group are implicated in a number of neurodegenerative diseases. One group of neurodegenerative diseases, taupathies, is associated with tau protein. A number of biophysical and spectroscopic studies have revealed that tau protein can expand to a largely extended state and to transition rapidly between many different conformations. Although many of the techniques that elucidate structure of macromolecules have provided important information about the folded states of proteins, important information about the transition between the extended conformation states remain obscure. The goal of our work in the first part is the development of a new methodology to detect the presence of substructures in tau protein. This methodological advance may provide new insights in understanding tau protein behavior and its tendency toward aggregation. In this research, atomic force microscopy (AFM) has been used to study the effects of heparin polyanions on the conformational dynamics of tau protein. In AFM measurements tau protein fragment (255-441) equipped with cysteine at the C terminus was attached to the gold-coated AFM probe. Experiments measure interaction between tau-protein equipped tip and various surfaces. Distances and forces of sudden ruptures are extracted from the recorded approach-withdraw dependencies. The experiments were performed with using various concentrations of 11 kDa heparin solution. In addition, results obtained for heparin with molar mass of 11 kDa are compared with results obtained for heparin with molar mass of 18 kDa. Our data show that, heparin affected tau protein in concentration-dependent manner. The effect of adding heparin at concentration of 10 ụM and above was in apparent decrease of the rupture distances and the interaction forces. Changes in behavior of tau protein molecules after adding heparin to solution were irreversible: removal of heparin did not restore behavior of tau protein molecules. The irreversible changes were also concentration dependent. Adding 18 kDa heparin to solution shows qualitatively similar changes to those observed in 11 kDa heparin but the changes are more pronounced. We suggest that the observed irreversibility might be caused by irreversible binding of heparin molecules to tau protein molecules such that heparin molecules remains bound even after removing heparin molecules from solution. In order to determine whether accumulation of heparin on the tip caused the irreversible changes, we designed another approach where the heparin molecules were grafted to the gold-coated surface to prevent heparin deposition on tau-protein-coated tip. Comparing the results measured with two different approaches revealed that the reduction in the rupture distance is observed with heparin grafted on the surface, but this decrease is reversible: rupture distance restores on surface that does not contain heparin. In the second part of this dissertation, a new method to analyze transitions that occur after rupture is employed. It is noted that transitions after a rupture event in a series of rupture events occurs considerably slower than the expected transition of freely moving AFM probe. To model such transitions a simple viscoelastic Kevin-Voigt model is used to fit the force transients after rupture events. This approach allows to extract viscous properties of molecules that remain attached to the AFM probe, and to see how the model parameters change with changes in the environment. Results indicate that after adding and removing heparin, transitions exhibit more elastic (less viscous) behavior. This observation is consistent with picture of somewhat compact structure of tau protein with bound heparin as obtained from the rupture distance analysis.

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