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PEDS Advance Access originally published online on August 28, 2008
Protein Engineering Design and Selection 2008 21(11):645-652; doi:10.1093/protein/gzn043
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Making a single-chain four-helix bundle for redox chemistry studies

Kristina Westerlund2, Sean D. Moran3, Heidi K. Privett2, Sam Hay2, Jüri Jarvet2, Brian R. Gibney3 and Cecilia Tommos1,2,4

1Department of Biochemistry and Biophysics, University of Pennsylvania, 905 Stellar-Chance Laboratories, Philadelphia, PA 19104-6059, USA 2Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University SE-106 91, Stockholm, Sweden 3Department of Chemistry, Columbia University, New York, NY 10027, USA

4 To whom correspondence should be addressed.E-mail: tommos{at}mail.med.upenn.edu

The construction and characteristics of the stable and well-structured {alpha}4W protein are described. The 117-residue, single-chain protein has a molecular weight of 13.1 kDa and is designed to fold into a four-helix bundle. Experimental characterization of the expressed and purified protein shows a 69.8 ± 0.8% helical content over a 5.5–10.0 pH range. The protein is thermostable with a TM > 355 K and has a free energy of unfolding as measured by chemical denaturation of –4.7 kcal mol–1 at 25°C and neutral pH. One-dimensional (1D) proton and 2D 15N-HSQC spectra show narrow, well-dispersed spectral lines consistent with a uniquely structured {alpha}-helical protein. Analytical ultracentrifugation and NMR data show that the protein is monomeric over a broad protein concentration range. The 324 nm emission maximum of the unique Trp-106 is consistent with a sequestered position of the aromatic residue. Additionally, differential pulse voltammetry characterization indicates an elevated peak potential for Trp-106 when the protein is folded (pH range 7.0–8.5) relative to partly unfolded (pH range 11.4–13.2). The oxidation of Trp-106 is coupled to proton release as shown by a 53 ± 3 mV/pH unit dependence of the peak potential over the 7.0–8.5 pH range.

Keywords: amino-acid radicals/four-helix bundle/NMR/protein design/Rop

Received April 9, 2008; revised July 2, 2008; accepted July 29, 2008.


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