The HP-1 maquette: from an apoprotein structure to a structured hemoprotein designed to promote redox-coupled proton exchange. Academic Article uri icon

abstract

  • Synthetic heme-binding four-alpha-helix bundles show promise as working model systems, maquettes, for understanding heme cofactor-protein assembly and function in oxidoreductases. Despite successful inclusion of several key functional elements of natural proteins into a family of heme protein maquettes, the lack of 3D structures, due principally to conformational heterogeneity, has prevented them from achieving their full potential. We report here the design and synthesis of HP-1, a disulfide-bridged two-alpha-helix peptide that self-assembles to form an antiparallel twofold symmetric diheme four-alpha-helix bundle protein with a stable conformation on the NMR time-scale. The HP-1 design strategy began with the x-ray crystal structure of the apomaquette L31M, an apomaquette derived from the structurally heterogeneous tetraheme-binding H10H24 prototype. L31M was functionally redesigned to accommodate two hemes ligated to histidines and to retain the strong coupling of heme oxidation-reduction to glutamate acid-base transitions and proton exchange that was characterized in molten globule predecessors. Heme insertion was modeled with angular constraints statistically derived from natural proteins, and the pattern of hydrophobic and hydrophilic residues on each helix was then altered to account for this large structural reorganization. The transition to structured holomaquette involved the alteration of 6 of 31 residues in each of the four identical helices and, unlike our earlier efforts, required no design intermediates. Oxidation-reduction of both hemes displays an unusually low midpoint potential (-248 mV vs. normal hydrogen electrode at pH 9.0), which is strongly coupled to proton binding, as designed.

published proceedings

  • Proc Natl Acad Sci U S A

altmetric score

  • 3

author list (cited authors)

  • Huang, S. S., Koder, R. L., Lewis, M., Wand, A. J., & Dutton, P. L.

citation count

  • 67

complete list of authors

  • Huang, Steve S||Koder, Ronald L||Lewis, Mitchell||Wand, A Joshua||Dutton, P Leslie

publication date

  • April 2004