Oakland University

Summer Institute in Bioengineering and Health Informatics 2007

Superoxide Dismutase Dimer

Investigating the Folding Properties of Superoxide Dismutase

 The focus of our research is to run parallel chemical and computational experiments to elucidate the folding properties of the protein, Superoxide Dismutase (SOD).  Our goal is to analyze the folding pattern of SOD using Fluorescence Resonance Energy Transfer (FRET) as well as develop a computer simulation of its folding and unfolding.

 

                 Kim and Nadia have been performing chemical experiments in the wet lab to prepare the protein for FRET analysis.  The first step was to strip the native protein of its metal ions, as they affect the light absorbance properties of the protein.  Dialysis was used to accomplish this by exchanging various buffers of different pH to disrupt the electrostatic salt bridges that hold the metal ions in place.  Due to osmotic pressure, they diffused away from the protein and were able to be removed by exchanging the dialysis fluids.  The Tyrosine (Tyr) residues of the apoprotein were then nitrated using organic synthesis, a necessary step for FRET analysis.  Nitration of Tyr creates a moiety that absorbs emitted fluorescence, whereas unmodified Tyr absorbs light and emits fluoresce.  Therefore, we will create a SOD protein dimer with one of its domains containing a nitrated Tyr residue, and its homologous domain containing an unmodified Tyr residue.  FRET will be used to determine the position of the Tyr residues relative to one another as the protein is denatured and refolded based on the absorbance of fluorescence by the modified Tyr (acceptor) and unmodified Tyr (donor).

 

Jose and Megan have been running a parallel computational approach to determine the folding pattern of SOD.  Megan has been working with Assisted Molecule Building with Energy Refinement  (AMBER).  This software relies on using the summation of the lowest energy conformation expenditures of the protein to determine and model how the protein folds and unfolds.  Jose has been developing a Monte Carlo program, a tool which utilizes randomization of moves as well as least energy conformations to predict the final folded structure of a protein in a 3D lattice model.  Also together, they have been working on developing a deterministic method to predict the protein folding pathway. The method uses set theory and simple mathematics as its basis.

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