Leucine zipper peptides provide simple model systems for studying both the intramolecular and intermolecular interactions that govern protein folding. The synthetic 33-residue peptide GCN4-p1, derived from the yeast transcriptional activator GCN4, forms a stable biomolecular coiled-coil structure [O'Shea, E. K., Klemm, J. D., Kim, P. S., & Alber, T. (1991) Science 254, 539-544]. The guanidine-HCl induced equilibrium unfolding of this peptide at 5 degrees C and pH 7.0 yields a standard state free energy of 10.49 +/- 0.23 kcal (mol dimer)-1 when fit to a two-state model involving the native dimer and the unfolded monomer. The unfolding and refolding kinetics of GCN4-p1 were monitored by stopped-flow circular dichroism spectroscopy as a function of both peptide concentration and final denaturant concentration. The unfolding kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding kinetics, which are dependent on both peptide and guanidine concentration, are well described by a simple bimolecular association reaction. A simultaneous fit of all of the unfolding and refolding kinetic data to the model, N2[symbol: see text]2U, yields refolding and unfolding rate constants in the absence of denaturant of 4.2 x 10(5) M-1 S-1 and 3.3 x 10(-3) S-1, respectively. The equilibrium unfolding curve is accurately predicted from these rate constants, providing further support for the validity of the two-state kinetic model.

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS)

A hybrid analysis that combines the maximum entropy method (MEM) with nonlinear least squares (NLS) fitting has been developed to interpret a general time-dependent signal. Data that include processes of opposite sign and a slow baseline drift can be inverted to obtain both a continuous distribution of lifetimes and a sum of discrete exponentials. Fits by discrete exponentials are performed with initial parameters determined from the distribution of lifetimes obtained with the MEM. The regularization of the parameter space achieved by the MEM stabilizes the introduction of each successive exponential in the NLS fits. This hybrid approach is particularly useful when fitting by a large number of exponentials. Revision of the MEM "prior" based on features in the data can improve the lifetime distribution obtained. Standard errors in the mean are estimated automatically for raw data. The results presented for simulated data and for fluorescence measurements of protein folding illustrate the utility and accuracy of the hybrid algorithm. Analysis of the folding of dihydrofolate reductase reveals six kinetic processes, one more than previously reported.

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Single tryptophan mutants of the trp aporepressor, tryptophan 19 + phenylalanine (W19F) and tryptophan 99 -phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-11s component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63,741-750).On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission. Thus, changes in the fluorescence properties of the two intrinsic tryptophan residues in trp aporepressor upon unfolding are explained readily in terms of disruption of the dimer interface.Keywords: mutagenesis; protein folding; trp aporepressor; tryptophan fluorescence Fluorescence spectroscopy offers a variety of static and dynamic properties that are useful for monitoring proteinunfolding reactions induced by heat, chemical denaturants, or pressure. This application relies on the sensitivity of intrinsic tryptophan and tyrosine residues to their immediate environment. Changes in local environment generally coincide with the global loss of secondary and tertiary structure because the unfolding transitions of many proteins are highly cooperative. Thus, the dependence of steady-state fluorescence intensity or emission energy on the concentration of the denaturant can provide values for the free energy of folding (Tanford, 1970;Pace, 1986 site-directed mutagenesis followed by examination of the 'Present address: Eli