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)
Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each 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 at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = 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 mM). When extrapolated to mu = O, k(on) was approximately 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 (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state 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 folding 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.
Little is known about the extent to which protein flexibility contributes to antigen-antibody recognition and cross-reactivity. Using short coil peptides (leucine zippers) as model antigens, we demonstrate that a monoclonal antibody can force a noncognate peptide into a conformation that is similar to the conformation of the cognate peptide against which the monoclonal antibody is directed. Monoclonal antibodies 29AB and 13AD were raised against the 29-residue peptide LZ (Ac-EYEALEKKLAALEAKLQALEKKLEALEHG-amide) that forms a very stable coiled coil. The two antibodies cross-reacted strongly with the random coil analogue LZ(7P14P) that contains Lys-->Pro and Ala-->Pro substitutions in positions 7 and 14, respectively. The antibody-bound peptide LZ(7P14P) adopted an altered conformation that possibly was coiled coil-like, as shown by CD difference spectroscopy and fluorescence quenching experiments on coumarin-labeled peptides. Isothermal titration calorimetry revealed that the cross-reaction of antibodies 13AD and 29AB with the random coil peptide LZ(7P14P) exhibited a large unfavorable entropy. This, however, was strongly compensated by a more favorable enthalpy, resulting in only a small difference between the association constants for peptide LZ and LZ(7P14P), respectively. To investigate the opposite type of cross-reaction, monoclonal antibody 42PF was raised against the random coil peptide LZ(7P14P). 42PF cross-reacted with coiled coil peptide LZ by forcing it to dissociate into single chains. Enthalpy/entropy compensation again enabled the cross-reaction, which now was entropically favored and enthalpically disfavored. The rate of reaction of antibody 42PF with peptide LZ was controlled by the rate of dissociation of LZ into single chains. This observation, as well as the generally much slower reaction rate with the noncognate peptides, indicated that the cross-reactivity occurred because the antibody selected the conformer of the antigen that binds the strongest, a mechanism we call "induced fit by conformational selection."
Leucine zippers are composed of amphipathic «-helices containing heptad repeats (abcdefg) in which hydrophobic residues are frequent at a and d. The motif is found in many DNAbinding proteins.* 1The variety of combinatorial interactions between different -helices provides for the control of function of basic-region leucine zipper transcription factors.2 Physical studies established that interhelical hydrophobic interactions between residues in the a and d positions as well as interand intrachain electrostatic interactions govern the formation and stability of homomeric and heteromeric leucine zippers.2•3 4The kinetics and mechanisms by which the separate chains of a leucine zipper assemble are not known. Here we show that the assembly of a dimeric leucine zipper involves conformational rearrangements after the initial association of chains.Residues 249-281 of the yeast transcriptional activator protein GCN4, named peptide GCN4-pl,4a form a dimeric, parallel coiled coil.4b•5 The helices are only stable when folded in the coiled coil conformation but not as individual monomeric peptide chains.6Therefore, it seems unlikely that the two unfolded chains (M) associate to the native leucine zipper dimer (D) in a single step. Rather, monomers (perhaps in a partially folded state) probably associate in a concentration-dependent reaction to a dimeric intermediate (D*), which then relaxes to the native dimer (D) via one or more monomolecular steps. A minimal model of the mechanism is + Z D* ¿ D(1)The two reaction steps are characterized by the relaxation times and 2.depends on the initial peptide concentration, while t2 is apparently concentration-independent unless the two steps are strongly coupled (ti r2).We synthesized peptide FLU-GGG-GCN4-pl corresponding to GCN4-pl with a fluorescein group (FLU) attached to the N-terminus via a triglycine spacer (GGG).7 Fluorescence emission of FLU-GGG-GCN4-pl is quenched, presumably through selfquenching of the two FLU groups in the parallel coiled coil dimer, when compared to the unfolded peptide in 8 M urea or to a • Author for correspondence: phone,
and ¶Institut fü r Polymere, ETH Zü rich, Switzerland Basic region helix-loop-helix (bHLH) transcription factors regulate key steps in early development by binding to regulatory DNA sites as heterodimers consisting of a tissue-specific factor and a widely expressed factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E47, to understand why these proteins preferentially associate in heterodimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric. Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in and around the loop of the E47 bHLH domain contribute to its markedly greater stability. An altered MyoD bHLH substituted with the loop segment from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation constants, despite the very different stabilities of these protein dimers off DNA. A 1:1 mixture of these bHLH domains forms almost exclusively heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions at the dimer interface. These findings suggest that preferential association of MyoD with E47 in DNA complexes results from more favorable DNA contacts made by one or both subunits of the heterodimer in comparison with either homodimeric complex.At the heart of transcriptional initiation are numerous cooperative interactions between basal factors, transcriptional activators, and their promoter DNA sites. Many activating transcription factors bind to their DNA targets as heterodimers in which each subunit has a distinctive DNA binding preference and transcriptional activity. The strength of transcriptional activation is modulated by the choice of subunits forming these protein dimers, their cellular concentrations, and their affinities for DNA regulatory sites. Basic region helix-loop-helix (bHLH)
and Macalester College, MinnesotaIt has long been recognized that a common problem in describing ecological or social psychological relationships can involve variables one of which is ranked, the other dichotomous. According to Whitfield (1947), and valid even a t a time of justified multivariate emphasis, '. . . in applied or field work the relation of psychological "measurement" and an external criterion nearly always appears in this form.' Subsequent to Whitfield's own modification of Kendall's tau, several other rank-biserial coefficients have been defined. In particular, Brogden (1949), Cureton (1956) and Glass (1966) have written expressions based on Spearman-type approaches. An interesting aspect came to light when Stanley (1968) reemphasized their essential equality and, moreover, proved them to be identical to a point-biserial r corrected by its maximum theoretical value.Empirical studies to date d o not show any very prominent use (or awareness) of the available coefficients. A contemporary neglect of correlation in favor of significance statistics, on which Bakan (1966) and others have commented with occasional dismay, may have contributed to this development. But also -with the probable exception of Glass' rb coefficient -several of the measures are either not very easily computed under field conditions or the computational apparatus seems out of proportion vis-l-vis the kinds of small samples with which many of us are so familiar. Be this as i t may, we feel that simplified solutions will make the techniques more accessible and may in fact promote a better understanding of the problem area itself. In this connection we note, first, that numerous significance tests utilizing ranked data exhaust the same maximal information (cf. Lienert, 1962). Second, many comparisons of central tendency, especially in the case of small N, are currently performed using the Wilcoxon T or the Mann-Whitney U-tests.Given the above rationale a rank correlation, which we propose to call rc, can be defined as rL. = 1 ---where Pu is the theoretical mean for the sample sizes in question, with U as usually defined being the smaller of the two cumulative countsU max ---2 -2 obtained via the Mann-Whitney procedure. Since in turn, pu =
The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCNCpl of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX,EAKX,QALEKKLEALEHC-amide:peptide LZ ( X , , X2 = Leu), peptide LZ(12A) (X, = Ala, X2 = Leu), and peptide LZ(16N) (X, = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two-and threestranded leucine zippers composed of chains from LZ and LZ(12A), or from GCNCpl and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.
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