Da. Moffet et al., Carbon monoxide binding by de novo heme proteins derived from designed combinatorial libraries, J AM CHEM S, 123(10), 2001, pp. 2109-2115
Carbon monoxide binding was studied in a collection of de novo heme protein
s derived from combinatorial libraries of sequences designed to fold into 4
-helix bundles. The design of the de novo sequences was based on the previo
usly reported "binary code" strategy, in which the patterning of polar and
nonpolar amino acids is specified explicitly, but the exact identities of t
he side chains are varied extensively.(1) The combinatorial mixture of amin
o acids included histidine and methionine, which ligate heme iron in natura
l proteins. However, no attempt was made to explicitly design a heme bindin
g site. Nonetheless, as reported previously, approximately half of the bina
ry code proteins bind heme.(2) This collection of novel heme proteins provi
des a unique opportunity for an unbiased assessment of the functional poten
tialities of heme proteins that have not been prejudiced either by explicit
design or by evolutionary selection. To assess the capabilities of the de
novo heme proteins to bind diatomic ligands, we measured the affinity for C
O, the kinetics of CO binding and release, and the resonance Raman spectra
of the CO complexes for eight de novo heme proteins from two combinatorial
libraries. The CO binding affinities for all eight proteins were similar to
that of myoglobin, with dissociation constants (K-d) in the low nanomolar
range. The CO association kinetics (k(on)) revealed that the heme environme
nt in all eight of the de novo proteins is partially buried, and the resona
nce Raman studies indicated that the local environment around the bound CO
is devoid of hydrogen-bonding groups. Overall, the CO binding properties of
the de novo heme proteins span a narrow range of values near the center of
the range observed for diverse families of natural heme proteins. The meas
ured properties of the de novo heme proteins can be considered as a "defaul
t" range for CO binding in alpha -helical proteins that have neither been d
esigned to bind heme or CO, nor subjected to genetic selections for heme or
CO binding.