| As part of a “Structural Proteomics Initiative” at the Ontario Cancer Institute, we have solved the three dimensional solution structure of a protein with no previously known function or structure. The protein is a 77 residue peptide from the archaebacterium Methanobacterium thermoautotrophicum ΔH called MtH895. Using a combination of computational tools and modern NMR spectroscopy, we show (in chapter two of this thesis) that the protein adopts a well-defined fold similar to a class of redox proteins called thioredoxins/glutaredoxins. More detailed analysis shows that even though this protein has a glutaredoxin-like structure it appear to function as a thioredoxin. Indeed it appears to be the smallest known thioredoxin yet identified.; In chapter 3 of this thesis, the expression, purification, 15 N and 13C/15N double labeling of another protein, MtH807, from the same archeon, is described. Several optimization protocols have been discussed that resulted in{09}a yield of ∼40mg/L of protein from rich media and ∼10mg/L from minimal media. More than 97% of the backbone amide resonances are visible in the 1H- 15N-HSQC spectrum. ∼25% of preliminary spin system assignments have been done. |
