During the past month, I’ve been meeting with Dr. Pastrana to discuss the theoretical background of two-dimensional spectroscopy (2D-COS), and its application to infrared. This instrument can provide a structural analysis of proteins under study, and can describe by means of a contour map representation, how their secondary structure is altered when subjected to different environmental perturbations. Although this technique has been used in conjunction with other spectroscopic techniques, our lab is currently interested in its use for 2D-IR spectral analyses.
Environmental perturbation studies can include pH gradients, temperature gradients, etc…
The way an experimental procedure is typically approached is described as follows: A series of perturbation-induced dynamic spectra are collected in a systematic manner, and then transformed into a set of 2D-correlation spectra by cross-correlation analysis (1). I look forward to using this instrument to test the hsfi1 domains I’ve been working on, as well as the hcentrin-hsfi1 complexes I’ve worked on as well. This data can then be compared with the circular dichroism experiments for comparison and/or supplemental structural characterization.
I’ve currently been working on the isolation of the hsfi1 domains 10-12 using a GST affinity column. Because we are trying to determine the most effective protocol purification for this protein, in conjunction with two more students, I’ve been purifying small protein pellet samples using variations in our current protocol, with hopes of determining which factors are contributing to the loss of large amounts of the protein throughout the several steps the purification procedure entails. I believe I’ve so far only completed 10 percent of what I’ve intended to work on for the semester. There is much to work on and learn from. Hsfi1 has shown to be very insoluble and difficult to handle. We expect to have a strong set of results
