De Novo Design of Proteins - Peptide and Protein Design for Biopharmaceutical Applications - page 209

stapling' as a means to stabilize a-helical peptides to target pro-
tein-protein interactions, including the enforced helicity in a BAD
BH3 that interacts with antiapoptotic BCL-X L [16,17]. The 'sta-
pling' was achieved by incorporation of an a,a-disubstituted
unnatural amino acid [18,19], an alkenyl Ala analogue with a
terminal double bond, placed in i and i þ 4or i þ 7 positions. The
'staple' was formed by ruthenium-catalysed olefin metathesis,
providing the cyclic peptide.
(iv) Addition of nonpeptide templates to initiate helix formation
[20].
(v) Introduction of charged residues near the amino or carboxyl
termini to electrostatically stabilize the helix [12,21-23] due to
the macrodipole, i.e. negatively-charged amino acid residues in
the first turn (N-terminus) and positively-charged ones in the
last turn (C-terminus) [24].
(vi) Introduction of 'caps' at the N- or C-termini to hydrogen bond
to the helix ends, thus stabilizing the helix [24-30]. In an
a-helix, the NH protons point toward the N-terminal while
the CO carbonyls point toward the C-terminal end of the
helix. This leaves 'unsatisfied' hydrogen-bond donors and
acceptors at the two termini. At the N-terminus of the helix,
the side chains of Asn, Ser, Asp or Thr residues can hydrogen
bond to exposed amide protons: for example, from the O d to a
backbone NH of residues i þ 3 (N3) or, less often, i þ 2(N2)
[24,25,31]. The residue preceding the N-cap is often apolar
and can form a hydrophobic interaction with the side chain
of the fourth helical residue, forming a hydrophobic
stable [32]. Also, the so-called 'SXXE box' (a sequence of
SerXxxXxxGlu), which provides hydrogen bonding between
the N-cap amide and the side chain of Glu or Gln at an i þ 3
position [31]. C-terminal caps include Gly in the a L conforma-
tion (as in a left-handed a-helix, which is not allowed for
L -amino acids; see Ramachandran plot), or a Pro residue two
residues from the C-terminus of the helix [33,34].
Interestingly, Pro in position N1 (first residue after the 'cap')
can be a helix-initiator.
(vii) Addition of hydrogen bonds between side chains one turn apart
to stabilize the helix [10,12].
(viii) Introduction of a hydrogen-bond surrogate by replacement of
one of the main-chain intramolecular hydrogen bonds with a
covalent linkage (for review, see [35]).