Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors, A parallel leucine zipper composed of the acidic chain X(1)-EYQALEKEVAQLEAENX(2)-ALEKEVAQLEHEG-amide and the basic chain X(1)-EYQALKKKVAQLKAKNX(2)ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and lo investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each b peptide alone did not form a leucine zipper at ionic strength (mu) <1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either as the N-terminus (X(1) = fluorescein-GGG, X(2) = Q) or in the center of the coiled coil (X(1) = acetyl, X(2) = W), Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer, The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction, Lowering of the ionic strength increased k(on) from 4 x 10(6) M(-1) s(-1) (mu = 525 mM) to about 5 x 10(7) M(-1) s(-1) (mu = 74 nM). When extrapolated to mu = 0, k(on) was similar to 10(9) M(-1) s(-1), which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM, Equilibrium association constants (K-a) of the heterodimeric zippers measured directly and calculated from kinetic constants (K-a = k(on)/k(off)) were in good agreement. The observed two-stale mechanism, the strong dependence on ionic strength of k(on) but not of k(off) and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent Golding to the final dimer is very rapid and follows a ''down-hill'' free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.