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Back to Dr. Waner's pageBiophysical Studies of Solution Phase Proteins
This area of research was developed during my work here at JCU. Work in these projects is done in collaboration with biochemists, such as Dave Mascotti at JCU. Currently our interests focus on some interesting behavior of the bacterial protein, Streptavidin (SA). This protein is widely used as a tool in numerous biotechnology applications. The major reason for its usefulness is the strong (Kd~10-15) and specific binding of biotin (vitamin H). Functionalization of a molecule or target of interest with either biotin or SA offers one the ability to design very specific assays, with low detection limits. The usefulness of SA is further enhanced by the structural stability of the tetramer, which is further enhanced by the binding of biotin. Despite its widespread use we have found a number of more fundamental questions about SA and its stability which have not been adequately answered in the literature. One issue that originally sparked our interest was that SA exhibits variability in its primary sequence, resulting from various proteolytic agents found naturally or introduced during the purification of commercial samples. This issue leads to commercial samples which exhibit varying heterogeneity.
Our first published work in this area relates to a study of the thermal stability in the presence and absence of sodium dodecyl sulfate (SDS). This study utilized polyacrylamide gel electrophoresis (PAGE), UV-vis and fluorescence spectroscopy, and differential scanning calorimetry (DSC) to develop a proposed model for the structural transitions SA undergoes.
We are continuing our work in this area to explore other open questions and to examine the effect of other denaturing agents on the thermal stability of SA.
Interested, want to know more? Email me.
References:
1. Waner, M.J., Navrotskaya, I., Bain, A. Oldham, E. D. and Mascotti, D. P., Thermal and sodium dodecylsulfate induced transitions of Streptavidin, Biophys. J., 2004, 83, 2701-2713.
2. Waner, M.J., Mascotti, D.P. A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein, J. Biochem. Biophys. Methods, 2008, 70, 873-877 (DOI 10.1016/j.jbbm.2007.06.001).
3. Mascotti, D.P.; Waner, M.J., Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory, J. Chem. Educ., 2010, 87 (7), 735–738. (DOI: 10.1021/ed100199j).