Making a single-chain four-helix bundle for redox chemistry studies
2008 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 21, no 11, 645-652 p.Article in journal (Refereed) Published
The construction and characteristics of the stable and well-structured alpha W-4 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 N-15-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.
Place, publisher, year, edition, pages
2008. Vol. 21, no 11, 645-652 p.
Natural Sciences Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:su:diva-57886DOI: 10.1093/protein/gzn043ISI: 000260144000002OAI: oai:DiVA.org:su-57886DiVA: diva2:418435
Projectsamino-acid radicals, four-helix bundle, NMR, protein design, Rop
authorCount :72011-05-232011-05-232011-05-23Bibliographically approved