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Histidine placement in de novo–designed heme proteins

Published online by Cambridge University Press:  01 September 1999

BRIAN R. GIBNEY  and
P. LESLIE DUTTON
BRIAN R. GIBNEY
Affiliation:
The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, B501 Richards Building, Philadelphia, Pennsylvania 19104
P. LESLIE DUTTON
Affiliation:
The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, B501 Richards Building, Philadelphia, Pennsylvania 19104
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Abstract

The effects of histidine residue placement in a de novo-designed four-α-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [{Ac-CGGGELWKL·HEELLKK·FEELLKL·HEERLKK·L-CONH2}2]2 composed of a generic (α-SS-α)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [{Ac-CGGGELWKL·AEELLKK·FEELLKL·AEERLKK·L-CONH2}2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10′ bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 15 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 μM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 μM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.

Keywords

de novo designelectrochemistryheme proteinmaquettemetalloprotein
Type
Research Article
Information
Protein Science , Volume 8 , Issue 9 , September 1999 , pp. 1888 - 1898
Copyright
© 1999 The Protein Society

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