C O M M U N I C A T I O N S
partners, while paired Asn side chains at a positions in a parallel
coiled-coil can H-bond to one another.7a In contrast, we find that
antiparallel Asn-Asn pairing is slightly more destabilizing than
antiparallel Asn/hydrophobic pairing. This result is consistent with
the low frequency of Asn in natural antiparallel coiled-coils.3,12c
(3) Parallel Ile-Val pairing is significantly more stabilizing (by ca.
50% in terms of ∆∆G) than is Ile-Ala,12a but we find that
antiparallel Ile-Val and Ile-Ala pairings are isoenergetic (both a/d
arrangements). The comparable stability of these pairings suggests
that the matching of â-branched residues with truncated side chains
may be especially beneficial in antiparallel coiled-coils, which is
consistent with the work of Oakley et al.14
Figure 2. (A) Helical wheel diagram showing the helical regions of
NT-C. (B) Partial helical net for NT-C. In each diagram, N-terminal segment
is shown in blue and the C-terminal segment is shown in red.
Table 1. Thermodynamic Data (∆Gcc)a Obtained from Thioester
Exchange of NT-C Mutants.
The results reported here suggest that the relationship between
the identity of paired core residues from partner R-helices and the
favorability of the helix-helix interaction varies significantly
between parallel and antiparallel orientations. These findings are
important because interfacial side-chain packing preferences are
likely to contribute to coiled-coil dimerization specificities in vivo,
but this aspect of helix-helix recognition is poorly understood at
present. The model system introduced here should be useful for
continued exploration of helix-helix recognition rules, including
evaluation of non-proteinogenic side chains.15
Ψ ) Leu
Ψ ) Ile
Ψ ) Val
Ψ ) Asn
Ψ ) Ala
X ) Leu
X )Ile
X )Val
X )Asn
X )Ala
-1.4
-1.7
-1.4
0.3
-1.3
-1.0
-0.8
0.2
-0.9
-0.8
-0.6
0.4
0.2
-0.1
0.0
0.5
0.4
-0.4
-0.9
-0.9
0.8
-0.7
-0.7
-0.5
0.0
a Values are reported in kcal/mol. Uncertainty ≈ (0.1 kcal/mol.
Table 1 shows ∆GCC values derived for NT-C and 24 mutants. Five
residues were examined at each site, Leu, Ile, Val, Asn, and Ala.
Our modular strategy required the synthesis of only 10 short
peptides to determine the 25 ∆GCC values. The broad trends among
these data are consistent with expectations based on extrapolation
from the large literature on parallel coiled-coils and from statistical
analysis of residue occurrence in natural antiparallel coiled-coils.1,12
Thus, the large aliphatic side chains of Leu and Ile participate in
the most stable intramolecular pairings, and introduction of a polar
Asn side chain causes a substantial loss of folded state stability.
These trends support our design hypothesis that the two 14-residue
segments engage in antiparallel coiled-coil formation in the full-
length thioesters (e.g., NT-C).
Acknowledgment. This research was supported by the NIH
Grant GM-61238. The authors would like to thank Darrell R.
McCaslin for assistance with the AU experiments.
Supporting Information Available: Experimental details, CD
analysis, and HPLC chromatograms. This material is available free of
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