Date of Award

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Latour, Robert A

Committee Member

Boland , Thomas

Committee Member

Powell , Gary L

Committee Member

Guiseppi-Elie , Anthony

Committee Member

Vertegel , Alexey

Abstract

Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for fabrication of stable, reusable substrates with a highly specific functionality. The performance of these systems is greatly influenced by the orientation and conformation of the immobilized enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces using circular dichroism (CD) spectropolarimetry and evaluate their bioactivity using colorimetric assays. When combined with knowledge of an enzyme's native structure, these methods provide a means to correlate changes in enzyme bioactivity post-adsorption with its adsorbed orientation and conformation. Using this approach, we investigated the adsorption behavior of a set of model enzymes [trypsin (TRP; 23.8 kDa), lysozyme (HEWL; 14.4 kDA), xylanase (XYL; 21.3 kDa), and glucose oxidase (GOx; 160 kDa)] on OH-, CH3-, NH2-, and COOH-terminated alkanethiol self-assembled monolayer (SAM) surfaces. The bioactivities of the small proteins, TRP, HEWL, and XYL, had pronounced variations between the different SAM surfaces despite their structural stability, highlighting the role of adsorbed orientation on bioactivity. In contrast, GOx, which is a much larger protein, exhibited wide variations in both its structure and bioactivity after adsorption, with adsorption-induced conformational changes actually enhancing its bioactivity. In order to gain further insights into adsorbed orientation and conformation, adsorbed HEWL and GOx layers on the various SAMs were chemically modified with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS), which selectively labels solvent accessible tryptophan residues. Analysis of tryptophans labeled when the protein is in solution vs. when adsorbed can provide insights into the orientation of the adsorbed protein and adsorption-induced changes in its tertiary structure. The ratio of modified tryptophans per HEWL molecule decreased on every surface in comparison to the free floating protein, indicating that these hydrophobic residues were interacting with the surface, rendering them solvent inaccessible. Furthermore, it suggests that the protein was not exposing new tryptophans to solution due to adsorption-induced unfolding. In contrast to HEWL, the number of modified tryptophans per GOx molecule increased after adsorption on the four SAMs, which clearly shows that the tertiary structure of GOx is significantly altered by adsorption. These results reinforce the CD data that GOx undergoes substantial conformational changes upon adsorption causing previously inaccessible residues to be solvent accessible post-adsorption. These results provide new insights into protein-surface interactions at the molecular level and demonstrate that adsorption can either promote or inhibit bioactivity depending on how the surface chemistry influences the orientation and conformational state of the protein on the surface.

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