Recently, results using electrospray ionization mass spectrometry (ESI) have shown that it is feasible to study protein-metal, protein-protein, and protein-DNA interactions. ESI is a soft ionization technique capable of transferring to the gas phase non-covalent complexes formed in solution. How well the nature of the interaction observed by electrospray reflects the nature of the condensed phase is still under investigation. We propose to study these interactions to establish the strengths and weaknesses of the method and compare it to other techniques. Initially we will investigate protein-metal interactions. The binding strength and specificity determined directly by mass spectrometry can be compared with other methods such as equilibrium dialysis, fluorescence spectroscopy and circular dichroism. Specifically, we will study the interactions between the prion protein (PrP) and copper (II) ions. The PrP has been shown to bind copper and its binding constant has been determined by other methods. By analyzing peptides that correspond to sections of the protein we can determine which area of the protein binds the metal most strongly and specifically. ESI will allow us to carefully monitor how a change in pH, ionic strength, type of metal, or concentration affects the interaction. With an established analytical method we can then study other interaction types. Specifically we wish to study the interaction between the estrogen receptor and DNA. It has been shown that estrogen receptor binds DNA specifically only in the presence of zinc, cadmium or cobalt. The DNA-binding domain binds copper metal more strongly, but when copper is bound DNA does not bind. As an extension of the above work we will first study the proteinmetal interaction before introducing DNA. We will then establish the analytical method to study the protein-DNA interaction using ESI. The ESI conditions used to study DNA differ from the conditions normally used for proteins, thus, satisfactory conditions for both will need to be determined. The effect that such a compromise has on the DNA-protein interaction will be assessed. The protein binds as a dimer, thus, we shall attempt to monitor complexes containing DNA, two protein molecules and two metal atoms. The dissociation constants determined by ESI can be compared with previous studies. We will establish if ESI can distinguish between specific DNA versus non-specific DNA binding. There are suggestions that DNA binding is impaired by oxidation of the receptor. We will determine whether zinc binding is impaired by oxidation. Future work will involve measuring previously unstudied interactions that are amenable to the technique. The sensitivity of ESI will allow study of interactions and measurement of dissociation constants where traditional techniques fail due to poor sensitivity.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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