title: Making a single-chain four-helix bundle for redox chemistry studies.
abstract: The construction and characteristics of the stable and well-structured alpha(4)W 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 T(M) > 355 K and has a free energy of unfolding as measured by chemical denaturation of -4.7 kcal mol(-1) at 25 degrees C and neutral pH. One-dimensional (1D) proton and 2D (15)N-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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, 905 Stellar-Chance Laboratories, Philadelphia, PA 19104-6059, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Westerlund
authors.forename: Sean D
authors.initials: SD
authors.lastname: Moran
authors.forename: Heidi K
authors.initials: HK
authors.lastname: Privett
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.forename: Jüri
authors.initials: J
authors.lastname: Jarvet
authors.forename: Brian R
authors.initials: BR
authors.lastname: Gibney
authors.forename: Cecilia
authors.initials: C
authors.lastname: Tommos