Histidine placement in de novo–designed heme proteins
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...
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Published in | Protein science Vol. 8; no. 9; pp. 1888 - 1898 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
Bristol
Cambridge University Press
01.09.1999
Cold Spring Harbor Laboratory Press |
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0961-8368 1469-896X |
DOI: | 10.1110/ps.8.9.1888 |