During Summer 2005 and 2006, I worked with a group of crop physiologists to monitor changes in overall plant health during the summer growing season. Growing up in a rural community meant that my first exposure to the scientific community was through agricultural. My two summers interning at Monsanto as part of their crop physiology team is clear extension of that initial interest. While at Monsanto, I worked with a team of scientists whose goal was to better understand the effects of gene mutations on several physiological traits in corn, cotton, and soybeans. As a crop physiology intern, my task was to monitor and help evaluate several different crop traits in the fields. To carry out this work, I used a modular photosynthesis tool called a LiCor(R). This tool allowed us to collect a variety of types of data relating to overall plant health. After the data was collected, it was analysed looking for overall trends in plant health to help other members of the team decide which construct to pursue in the future.

As part of my undergraduate research at Knox College (2009, BA in Biochemistry (with Honors in Chemistry)), I worked with Dr. Larry Welch and Dr. Andrew Mehl developing a fluorescence anisotropy based method to monitor protein folding dynamics via the Perrin equation in vitro.

Fluorescence anisotropy is a powerful fluorescence based spectroscopic technique which allow users to monitor changes in plane polarized light as it relates to a fluorophore or a fluorophore contain biomolecule. The concept of polarization stems a basic first principle of fluorescence which is that during the excitation of ground state electrons only those molecules whose dipole moments are at that moment aligned with incident light are excited.

Under normal conditions we don’t restrict the orientation (or polarization) of the light coming into the sample. However, fluorescence anisotropy allows the user to filter away all but a single orientation of flight, and therefore only those molecules whose orientation is aligned with incoming light are excited. During the excited state (i.e the fluorescence lifetime) some or all of that rotation may be retained by the fluorophore. These difference are what we measure during fluorescence anisotropy experiments.

In our case we hoped to use a modified version of the Perrin equation as means to determine the molecular volume of a given protein in solution, and then eventually monitor protein folding dynamics. Our idea was to use the tryptophan residue as a sort of ‘reporter fluorophore’ for detecting changes in a proteins structural motifs. Initially we undertook several proof concept molecules (fluorescein, L-Tryptophan, glucagon, and subtilisin carlsberg) as way to see if we could accurately calculate molecular volume of a known set of standards. Based on our initial set of experimental parameters it was apparent that our methodology worked well for monitoring changes only in cases where the fluorophore or the fluorescent residue was solvent facing. Current work in the Welch is still focused on developing methodologies for better use fluorescence anisotropy to determine protein dynamics.

With that information in mind we decided to start to investigate GrpE, a bacterial heat shock protein. We thought that GrpE would be a good model to start with because it lacks any native fluorescent amino acids which would make spectral deconvolution much more difficult. Furthermore, GrpE has several distinct independent secondary structures which could also be studied in the future, so of which had already been already isolated by previous members of the Mehl lab.

With the help of Acerrlys Discovery Pro (V. 2.1) we identified two possible sites of point mutation (V109W and T145W) which seemed unlikely to cause majors changes in either the protein’s secondary or tertiary structure. Furthermore, both sites seems to have a relatively high amount of solvent exposure making them good choices for our current methodology. However, expression (specifically amplification) and purification of these point mutants proved problematic in our hands, Mehl group members are currently working to develop better expression and purification methods for these GrpE point mutants.