VERY RAPID, IONIC STRENGTH-DEPENDENT ASSOCIATION AND FOLDING OF A HETERODIMERIC LEUCINE-ZIPPER

Leucine zippers (coiled coils) are dimerization motifs found in severa l DNA-binding transcription factors, A parallel leucine zipper compose d of the acidic chain X(1)-EYQALEKEVAQLEAENX(2)-ALEKEVAQLEHEG-amide an d the basic chain X(1)-EYQALKKKVAQLKAKNX(2)ALKKKVAQLKHKG-amide was des igned to study the kinetics of folding of a heterodimeric leucine zipp er and lo investigate the role of electrostatic attraction between opp ositely charged peptide chains to the folding reaction. Each b peptide alone did not form a leucine zipper at ionic strength (mu) <1 M becau se of electrostatic repulsion between like charges in a homodimer. The refore, the formation of the heterodimeric leucine zipper could be inv estigated by simple mixing of acidic and basic chains. To monitor fold ing, 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 r eaction 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 fluoresce nce 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) wa s 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 stren gth; k(off) decreased only by about 2-fold when mu was lowered from 52 5 to 74 mM, Equilibrium association constants (K-a) of the heterodimer ic zippers measured directly and calculated from kinetic constants (K- a = k(on)/k(off)) were in good agreement. The observed two-stale mecha nism, the strong dependence on ionic strength of k(on) but not of k(of f) and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an elec trostatically 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 electrosta tics to the stability of the folded leucine zipper.