X-ray diffraction from protein crystals remains the most reliable way to determine the molecular structure of proteins, and how this structure relates to biological function. However, we still lack the ability to predict solution conditions that support the nucleation and growth of high-quality protein crystals for X-ray diffraction studies. The overall goal of this thesis is two-fold: (a) determine the nucleation behavior and solubilities for lysozyme crystals with two distinct crystal structures (orthorhombic vs. tetragonal) and (b) investigate whether these changes in crystal habit and crystal solubility correlate with any discontinuities in the liquid-liquid phase boundary of lysozyme that occurs under the same solution conditions.
We measured lysozyme crystal solubility by nucleating and subsequently dissolving very small lysozyme crystals in highly supersaturated solutions. The presence of crystals in our samples is detected and monitored by measuring the light scattered off the micron-sized crystals. These "turbidity measurements" are repeated across a range of protein concentrations, for pH 4.6 and 5.6, thereby yielding the crystal solubility boundary. Changes in crystal structure are assessed at the end of the experiments by microscopic inspection of the distinct crystal habits.
Attractive protein interactions in solution also induce liquid-liquid phase separation. Similar to the crystal solubility measurement, we use the turbidity increase associated with liquid-liquid phase separation to map out this phase boundary. Since both crystal formation and liquid-liquid phase separation are driven by attractive protein interactions, we investigated whether the dramatic changes in crystal solubility associated with different protein crystal structures lead to any discernable “discontinuities” in the liquid-liquid phase boundaries.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-1122 |
| Date | 05 April 2008 |
| Creators | Apgar, Marc C |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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