Biomedical Engineering Reference
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H
b
H
b
and F
b
F
b
was strongly favoured over heterodimerization, which
indicated a potential for orthogonality between fluorous and hydropho-
bic interactions in protein design.
Marsh and coworkers designed a 27-residue peptide intended to form a
4-helix bundle-like structure with an antiparallel topology (Ac-GN
ADELYKE LEDLQER LRKLRKK LRSG NH
2
, a
4
-H) [86,109].
Stepwise substitution of Leu for hFLeu provided a
4
-F
2
, a
4
-F
4
and a
4
-F
6
,
in which 2, 4 and 6 layers, respectively, of the hydrophobic core were
repacked with hFLeu. CD spectroscopy revealed for all fluoroproteins an
extensively a-helical secondary structure. At high concentrations (up to
500 mM) all structures occurred predominantly as the tetramers, while at
lower concentrations monomer-tetramer equilibria were observed.
Apparently, the bigger hFLeu side chains could be accommodated
in the core. The stability of the protein structures increased with increas-
ing numbers of fluorinated side chains in the core. In addition, the
core of a
4
-F
6
appeared to be less conformationally dynamic than that
of a
4
-H.
Interestingly, a
4
-F
6
proved more stable to proteolysis than a
4
-H.
Surprisingly, TFE did not preferentially dissociate a
4
-F
6
. In contrast to
the observations by Kumar and coworkers, Marsh and coworkers
reported that they did observe indications for interactions between
a
4
-F
6
and a
4
-H, thus did not observe self-segregation in this protein-like
assembly. They speculated that in the Kumar peptide, not only fluorous
interactions but also steric effects from hFLeu, which is larger than Leu,
played a role.
Koksch and coworkers [110] used as a model system a 41-mer, RLEEL
REKLE SLRKK LACLK YELRK LEYEL KKLEY ELSSL E, which could
form an a-helical coiled coil as an antiparallel homodimer. In this
sequence, positions
a
and
d
had alkyl side chains, while
e
and
g
had
charged residues capable of forming interhelical salt bridges. Koksch
and coworkers studied the effect of mutating Lys
8
and Leu
9
with fluori-
nated analogues. They employed non-natural building blocks with
increasing degrees of fluorination (
S
)-ethylglycine (EGly), (
S
)-4,4-
difluoroethylglycine (DfeGly), (
S
)-4,4,4-trifluoroethylglycine (TfeGly),
(
S
)-4,4-difluoropropylglycine (DfpGly). Assuming that the trifluoro-
methyl moiety has approximately the size of an isopropyl group, Leu
would have space-filling properties as (
S
)-4,4,4-trifluoroethylglycine
(TfeGly). They evaluated the effects of introduction of fluorinated ana-
logues by two different methods: first thermodynamic stability and second
the ability to 'self-replicate' by the reaction of two peptide segments
through native chemical ligation to yield the full sequence. Substitutions
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