If you bother, it will talk to you

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

Summer’s gone

Throughout this past summer, I spent nine weeks conducting biostructural research under the guidance of Doctor Walter J. Chazin, from the biochemistry and biophysics’ faculty at Vanderbilt University, TN. His research interests seek in underlying molecular basis for biological specificity and biochemical funtion of proteins and nucleic acids using NMR spectroscopy, X-ray chrystallography, and other biophysical methods to characterize molecular structure and dynamics.

My project involved the binding affinity quantification of the 70N domain of Replication Protein A (RPA). RPA is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. It plays a central role in chromosomal DNA replication, repair and recombination pathways, protecting ssDNA from degradation by nucleases.  RPA also mediates interactions with specific proteins active in these various DNA processing events. This multi-functionality correlates with RPA’s modular structure.

RPA has three subunits, each named after its molecular weight:  RPA70 (domains N, A, B and C), RPA32 (domains N, D and C) and RPA14 (single domain).  The N-terminal domain of RPA70 (70N) is flexibly linked by an 80 amino acid linker to the rest of RPA 70.  It has long been established that RPA binds ssDNA with nM affinity through the action of domains 70A, 70B, 70C and 32D using 3 modes of binding. Tandem DNA binding by domains 70A and 70B  is required for high affinity.

Recently, a proposal has been made that 70N contributes to DNA binding function.  However, 70N binding affinity is more than 1000-fold weaker than RPA70AB and all evidence shows 70N is primarily a protein-protein interaction domain targeting transcription factors and checkpoint proteins such as p53 and ATRIP, respectively.  This study aimed to resolve this controversy by analyzing the effect of 70N on the ssDNA binding activity of the high affinity RPA DNA binding domains, 70A and 70B. We proposed the use of size exclusion chromatography, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC) to do a systematic comparison of the DNA binding properties of 70AB versus a 70NAB construct. We expect to show that 70AB DNA binding affinity is the same as 70NAB. This will provide conclusive evidence that the 70N domain is not involved in binding ssDNA.

For the course of this academic term, I will be in charge of isolating several domains of the hsfi1 protein. We have previously endeavored purifying this protein using several chromatographic techniques. After many unsuccessful trials using nickel columns for histidine-tagged hsfi1 proteins, we were finally able to isolate and sequence the hsfi1 protein. This isolated protein was a GST-tagged hsfi1 construct. Nonetheless, we were never able to isolate hsfi1 again. A recent, successful expression of both, hcentrin and hsfi1 proteins were obtained from a single protein batch pellet . It is thought hcentrin protein aids in hsfi1 solubility. We attribute our previous unsuccessful attempts in purifying hsfi1 to the single over expression of this protein absent of hcentrin. We believe the recovery of hsfi1 following the same previous protocols used for its purification will yield larger ammounts of the protein.  I also hope to repeat my circuar dichroism experiments of my hsfi1 and hcentrin constructs and complexes for secondary structure characterization. Due to my previous summer experience using ITC instruments for thermodynamic characterization of protein complexes formed in solution, I also hope to quantify the binding affinities of hcentrin and hsfi1 proteins.  One of my goals for this term and the next would be to  crystallize the hcentrin-hsfi1 complex. It’s been difficult to crystallize either one of these isolated proteins. Structural stability of the hcentrin-hsfi1 protein complex should yield more promising crystals that can diffract and allow its structural topology to be characterized.

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