| This dissertation describes the creation of a system to provide insight about the affects of protein expression on intracellular diffusion. Fluorescence recovery after photobleaching (FRAP) is used to obtain diffusion coefficients. Nuclear magnetic resonance spectroscopy detection of 19F isotopic-enriched proteins and fluorescent labeling with FlAsH and were attemped before settling on FRAP studies with green fluorecent protein (GFP). To regulate protein levels in Escherichia coli, several vector and promoter combinations were tested. Eventually, a single vector was created containing the structural gene for GFP under the lac promoter and a test protein under the araBAD promoter. With this vector, the test protein was expressed at varying levels and GFP was expressed at a constant level. Although chymotrypsin inhibitor 2 was originally chosen as the test protein, it was quickly replaced by alpha-synuclein, maltose binding protein, tau-40, and calmodulin. My most important result is that regardless of the type or amount of protein that was co-expressed, the GFP diffusion coefficient remained constant. We conclude that expression of these soluble proteins has little to no effect on the diffusion of GFP.;Several disadvantages of FRAP became apparent in the process of obtaining these data. FRAP and the other common techniques for measuring translational diffusion, present numerous obstacles when working with structures that are only slightly larger than optical resolution, such as Escherichia coli cells. To overcome these obstacles, I also developed a new method to assess diffusion using through-prism total internal reflection fluorescence microscopy with continuous photobleaching. Here, the theoretical basis for this technique is presented. I demonstrate its applicability by measuring the diffusion coefficient, 6.3 +/- 1.1 mum2/sec, of GFP in Escherichia coli cells. |
