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

The construction and characteristics of the stable and well-structured α4W 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 α-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. © The Author 2008. Published by Oxford University Press. All rights reserved.