De Novo Design of Proteins - Peptide and Protein Design for Biopharmaceutical Applications - page 222

charge repulsion if the dimerization is parallel. a 2 B was stable but
appeared to have adopted a molten globule-like conformation. Starting
from a 2 B, DeGrado and coworkers first designed a 2 C, in which half of the
Leu residues were substituted with aromatic and b-branched apolar side
chains [81]. However, only after substituting three of the interfacial apolar
residues for polar ones, providing the sequence a 2 D, did dimerization of
this sequence lead to a native-like protein structure [82]. Actually, these
mutations were originally intended to introduce a metal-ion binding site
into a 2 C. However, dimeric a 2 D also showed native-like character even in
the absence of metal ions, i.e. a 2 D had characteristics of folded proteins.
Remarkably, a 2 D had a 'bisecting U' folding topology, with diagonal
crossover loops and two parallel and two antiparallel helices [83].
Dimeric a 2 D was thermodynamically less stable than a 2 B, which indicated
that the native-like character of a 2 D was obtained by negative design,
which destabilized alternatively-folded states. In negative design, a struc-
tural feature is built into the sequence that will disfavour certain folds,
rather than directly favouring a topology (as for example the introduction
of a salt bridge might).
Schafmeister, Stroud and coworkers have expressed a de novo -
designed protein of 108 AA which folds as 4-helix bundles, as determined
by X-ray crystallography [84]. The protein, termed DHP 1 , was based on
the amphipathic, a-helical 24 AA sequence PD 1 [85].
Betz and DeGrado, and then Marsh and coworkers, have concluded
that antiparallel and parallel bundles differ in that the former are more
conformationally stable toward changes in the hydrophobic core, as subtle
changes in sequence of the latter or in buffer conditions can readily change
the oligomerization state between dimer, trimer and tetramer [86,87].
Baltzer and coworkers have in a series of papers described the design
and utilization of a 42-residue helix-loop-helix peptide designed to
dimerize [88-90]. For example, they reported the site-selective chemical
acylation of Lys and Orn residues. They were able to incorporate an
amino acid carrying a triaza-cyclononane that allowed binding of Zn(II),
which showed some transesterification activity.
6.3.3.3 a-helical metalloproteins
Once some of the first rules for de novo design of protein-like structures
with a-helices were established, work began to incorporate metal-ion
binding sites into de novo -designed proteins. The efforts by Pecoraro
and coworkers to this end have already been discussed above. Haehnel