32 KCALÆMOL)1), WITH ONLY A VERY SMALL ENTHALPIC CON-EITHER THE CJUN...

5.32 kcalÆmol

⁾¹

), with only a very small enthalpic con-

either the cJun or cFos target peptide. This structure is

maintained by core hydrophobic interactions, primarily

tribution (DH; ) 0.82 kcalÆmol

⁾¹

) to binding at 293 K.

The favourable entropy term arises mainly from desol-

brought about by knobs into holes packing between

a–a¢ and d–d¢ residues, and from which the bulk of stabil-

vation effects which outweigh the unfavourable confor-

mational penalty. This is consistent with an observed

ity arises. In addition, flanking electrostatic interactions

between g–e¢+1 core flanking residues are speculated to

weak enthalpic contribution to the free energy of bind-

play a primary role in specificity [22,23]. Together, both

ing. Indeed, the free energy of binding is 2–3 kcalÆmol

⁾¹

less than any of the antagonist–cJun or antagonist–

of these types of interaction are predicted to give rise to a

cFos complexes. ITC data collected from the leucine

favourable enthalpic transition upon binding. By con-

zipper region of cJun and cFos correlate poorly with

trast, the entropic term is largely dominated by the net

result of two opposing forces. The first, conformational

the findings of Seldeen et al. [18] (see Tables 1 and 2).

We believe that their data overestimate the free energy

entropy (DS

_conf

) results in a positive (unfavourable) net

of binding for the leucine zipper region in the absence

contribution to the overall free energy of binding. DS

_conf

arises from a reduction in conformational degrees of free-

of DNA. One possibility could be the use of a fusion

dom of backbone and side chain atoms as the molecule

construct with a (His)

₆

-tag and Trx-tag included to

necessitate purification and solubility of the cJun ⁄ cFos

folds and gains structure. By contrast, desolvational

entropy (DS

_solv

) contributes favourably to the net free

leucine zippers. Seldeen et al. noted that these addi-

energy of binding and results from the release of water

tional units were not anticipated to interact with the

bZIP domains of Jun and Fos.

molecules bound to regions of the target and antagonist

Our ITC data on the stability of the cJun–cFos

that become buried in fully formed complex.

interaction correlate well with thermal melting data

(see Table 2 and [11]), chemical denaturation data [12]

Wild-type Jun–Fos leucine zipper region

and earlier studies that have probed these regions [11]

(and references therein). In addition, both the bZIP

The native coiled coil region of this human transcrip-

tional regulator produces a relatively weak interaction,

coiled coil prediction algorithm and the base-optimized

weights method of in silico coiled coil stability predic-

as has been well documented [11,12,21]. Addition of

DNA and other factors such as disulfide bridges

tion anticipate the measured stability of all of our

[24,25] and additional flanking regions [18,26–28] have

coiled coils pairs with reasonable accuracy, giving us

confidence in the reliability of our data. In addition,

been shown to increase the stability of the complex. In

Table 3. Helical calculations to assist in establishing whether thepeptide is representative of a coiled coil structure [30–32].Averaged helicityin % predicted byFractionPeptides