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such that position 1 is taken from sequence A2, while position 9
matches the substitutions incorporated into sequences A4 and A5. An
additional sequence, B3, is formulated as a combination of sequence A3
and the position 9 substitution of histidine to tryptophan as taken from
control sequence X2. Each of the three designed sequences demon-
strates significant increases in fold stability relative to the original
compstatin sequence (table 2.2).
Another set of two additional sequences was identified with the
only difference between them being the specification of the residue at
position 4. For sequence C1, tyrosine was assigned to position 4, while
sequence C2 was selected to have valine at this position. For both
sequences, threonine was specified at positions 9 and 11, while posi-
tions 1 and 13 were set to isoleucine and valine, respectively. The choice
of isoleucine for position 1 helps to reduce the number of simultaneous
changes from the parent peptide sequence.
For both sequences C1 and C2 the stability calculations indicate a
substantial decrease in stability when compared to the parent peptide
sequence. Nevertheless, between sequences C1 and C2 there is strong
evidence for the preference of tyrosine at position 4. This prompted
closer examination of the residue selections at positions 9 and 11, the
two remaining positions not involved in the hydrophobic clustering of
compstatin. In particular, the specification of threonine at both posi-
tions 9 and 11 results in a negative net charge balance due to the
aspartate at position 6, especially because of the replacement of argi-
nine by threonine at position 11. This validates further the placement of
arginine at position 11 for the previous set of sequences (table 2.2).
The final set of sequences was designed in accordance with addi-
tional reductions in the number of simultaneous mutations relative
to the parent peptide sequence. Specifically, sequences D1 and D2
resemble sequences B1 and B2 with threonine instead of valine as the
C-terminal residue, a specification matching the composition of the
original parent peptide sequence. Both sequences provide significant
increases in fold stability. For sequences D1 and D2 the differences with
respect to the parent peptide sequence are isolated to the residue before
and after the b turn. Both the position 4 tyrosine and position 9 pheny-
lalanine substitutions provide enhancements to the fold stability of the
compstatin structure, and represent unforeseen and unpredictable
enhancements over the parent peptide sequence (table 2.2).
Experimental Validation
A number of the designed sequences presented above were constructed
and tested experimentally for their activity, without performing
NMR-based structural analyses. Since the ultimate goal is to enhance
the functional activity of compstatin, such achievements must be com-
plemented and verified through experimental studies. Rather than
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