Facile chemical synthesis and equilibrium unfolding properties of CopG.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Wales, TE; Richardson, JS; Fitzgerald, MC

Published Date

  • July 1, 2004

Published In

Volume / Issue

  • 13 / 7

Start / End Page

  • 1918 - 1926

PubMed ID

  • 15169951

Pubmed Central ID

  • 15169951

Electronic International Standard Serial Number (EISSN)

  • 1469-896X

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.04671804

Language

  • eng