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, DeltaG(f) and m-values of -13.42 +/- 0.04 kcal/mole dimer and 1.92 +/- 0.01 kcal/(mole M) were calculated for CopG.
Wales
Thomas E
TE
Department of Chemistry, Box 90346, Duke University, Durham, NC 27708-0346, USA.
Richardson
Jane S
JS
Fitzgerald
Michael C
MC
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D007700:Kinetics; D017510:Protein Folding; D013291:Streptococcus / Q000737:chemistry*; D013816:Thermodynamics; D014157:Transcription Factors / Q000138:chemical synthesis* / Q000737:chemistry*
D014157:Transcription Factors