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Published online before print May 28, 2004, 10.1110/ps.04671804
Protein Science (2004), 13:1918-1926. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Facile chemical synthesis and equilibrium unfolding properties of CopG

Thomas E. Wales1, Jane S. Richardson2 and Michael C. Fitzgerald1

1 Department of Chemistry, Duke University, Durham, North Carolina, 27708, USA
2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, 27710, USA

(RECEIVED January 30, 2004; FINAL REVISION March 30, 2004; ACCEPTED March 30, 2004)



Abstract

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, {Delta}Gf and m-values of –13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG.

Keywords: protein folding/unfolding; total chemical synthesis; protein design

Reprint requests to: Michael C. Fitzgerald, Department of Chemistry, Box 90346, Duke University, Durham, NC 27708-0346, USA; e-mail: michael.c.fitzgerald{at}duke.edu; fax: (919) 660-1605.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04671804.


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M. Wang, T. E. Wales, and M. C. Fitzgerald
Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold
PNAS, February 21, 2006; 103(8): 2600 - 2604.
[Abstract] [Full Text] [PDF]


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