Purification and Characterization of TRI 05 I13S M6I

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and redox potentials that can be useful in more complex systems. The use of (2,2′-bipyridin-5yl)alanine (Bpy-ala) as a Noncanonical Amino Acid (NCAA) has allowed Bpy to be incorporated into an amino acid sequence which can now function in a protein scaffold. Previous studies have utilized that power of Bpy-ala to design a protein that can assemble a homotrimeric protein complex in the presence of a divalent metal. However, the issue with this design was that when the homotrimer was formed and the divalent was removed, the protein complex would not dissemble indicating that it was not metal dependent. Point mutations were made to disrupt the protein-protein interactions to favor disassembly in the absence of a divalent metal. Successfully, a mutation was made that allowed the designed protein to be metal dependent for self-assembly. Nevertheless, an issue with this design is that it poorly incorporated ruthenium(II) into the tris Bpy complex forming [Ru(Bpy-ala)¬3]+2, which was one of the main goals of the original design. This thesis sets out to form TRI 05 I13S M6I which should uphold the same metal-dependence as its predecessor and should combine ruthenium (II) into the protein complex forming [Ru(Bpy-ala)¬3]+2. The thesis shows the success of formation and expression of TRI 05 I13S M6I in Escherichia coli cells. This thesis also reports several purification steps and procedures to not only purify TRI 05 I13S M6I but also removing both the His-tag sequence and Fe(II) from the protein. The thesis also shows that TRI 05 I13S M6I does not behave like its predecessor in that it is not metal dependent for self-assembly. While this may be true, this paper also reports the incorporation of ruthenium (II) in the protein structure. Though this may be the first time that ruthenium (II) has been recorded to be in the TRI 05 protein complex with a significant signal, it is still nowhere near the optimal fluorescence that small molecule Bpy can achieve by itself. The thesis reports potential conditions and a plan of attack that should drive this project forward into achieving an optimal signal of the [Ru(Bpy-ala)¬3]+2 complex in a TRI 05 protein scaffold.