Facile chemical synthesis and equilibrium unfolding properties of CopG

The 45‐amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc‐chemistry. The product obtained from the synthesis was readily purified by reversed‐phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three‐dimensional structures similar to the wild‐type protein prepared using conventional recombinant methods as judged by far UV‐CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride‐induced equilibrium unfolding properties of the wild‐type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two‐state process involving folded dimer and unfolded monomer. Using this model, Δ G f and m ‐values of −13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG.