Kinetics of Folding of Leucine Zipper Domains

Abstract

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils

“…CSL9C was shown spectroscopically to have similar behavior to TRI L9C with regard to affinity, pH-dependence, and geometry of metal binding. These results corroborated previous solution studies of Coil Ser derivatives suggesting that parallel aggregates were formed at higher pH values (34). In this study, we are using CSL9C for structural studies of the arsenic-bound peptide because it has proven more facile to form diffraction-quality crystals in comparison to the TRI peptides.…”

Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif

“…Of these proteins, the parallel dimeric coiled coil appears to be one of the best understood (11)(12)(13)(14)(15)(16), as this protein contains only ␣-helical elements of secondary structure. Indeed, a wealth of reports have been published on the folding of the yeast transcription factor GCN4-p1 (12,14,(17)(18)(19)(20)(21) with many reporting the protein to fold in a two-state mechanism.…”

Kinetics of Folding of the Myosin Rod

“…These results are compared with those reported recently for smaller coiled coils (Mo et al, 1991a,b;Wendt et al, 1995;Zitzewitz et al, 1995) and their biological relevance is discussed.…”

“…The rate-limiting step in the refolding of the myosin rod was slower than that observed for the corresponding transformation of small coiled-coil peptides (Wendt et al, 1995;Zitzewitz et al, 1995) or tropomyosin (Mo et al, 1991 a,b). This result is not suprising because the rate of folding of some oligomeric proteins may depend on their size and the presence of multiple structural domains (Milla and Sauer, 1994).…”

“…Interacting helical stretches propagate along the molecule and after small adjustenients in registration, the final coiled-coil dimer is formed. Even though simple monophasic kinetics have been found in the unfolding and refolding of this peptide (Zitzewitz et al, 1995), biphasic kinetics have been observed in the refolding of a small coiled-coil protein, tropomyosin (Mo et al, 1991 a, b) or other leucine-zipper peptides (Wendt et al, 1995). In these latter cases, the authors suggested Scheme 2b with the formation of the intermediate J,.…”