Reversible Denaturation Research Articles

Reversible Thermal Denaturation of Human FGF-1 Induced by Low Concentrations of Guanidine Hydrochloride

Human acidic fibroblast growth factor (FGF-1) is a powerful mitogen and angiogenic factor with an apparent melting temperature (Tm) in the physiological range. FGF-1 is an example of a protein that is regulated, in part, by stability-based mechanisms. For example, the low Tm of FGF-1 has been postulated to play an important role in the unusual endoplasmic reticulum-independent secretion of this growth factor. Despite the close relationship between function and stability, accurate thermodynamic parameters of unfolding for FGF-1 have been unavailable, presumably due to effects of irreversible thermal denaturation. Here we report the determination of thermodynamic parameters of unfolding (ΔH, ΔG, and ΔCp) for FGF-1 using differential scanning calorimetry (DSC). The thermal denaturation is demonstrated to be two-state and reversible upon the addition of low concentrations of added guanidine hydrochloride (GuHCl). ΔG values from the DSC studies are in excellent agreement with values from isothermal GuHCl denaturation monitored by fluorescence and circular dichroism (CD) spectroscopy. Furthermore, the results indicate that irreversible denaturation is closely associated with the formation of an unfolding intermediate. GuHCl appears to promote reversible two-state denaturation by initially preventing aggregation of this unfolding intermediate, and at subsequently higher concentrations, by preventing formation of the intermediate.

Stabilities of uncomplemented and complemented M15 beta-galactosidase (Escherichia coli) and the relationship to alpha-complementation.

M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C--uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein). Alpha-complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 beta-galactosidase. Alpha-complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation.

Characterization of the Transcriptional Activator CBF1 fromArabidopsis thaliana: EVIDENCE FOR COLD DENATURATION IN REGIONS OUTSIDE OF THE DNA BINDING DOMAIN

A transcriptional activator, CBF1, from Arabidopsis thaliana, which has the AP2 domain for DNA binding and regulates the cold acclimation response, was overexpressed in Escherichia coli, purified, and characterized. Analyses of the interaction between CBF1 and the C-repeat/dehydration-responsive element by fluorescence measurement showed that CBF1 binds to C-repeat/dehydration-responsive element as a monomer irrespective of the temperature. CD spectra of the intact and truncated CBF1 proteins (1-213, 41-213, 41-157, and 41-146) were measured to examine the temperature-dependent changes of the secondary structure of CBF1. The results suggested that the CBF1 protein has regions exhibiting reversible cold denaturation in the range between 30 and -5 degrees C and also has a region exhibiting thermal denaturation between 40 and 60 degrees C. This cold denaturation occurred in both the N-terminal and acidic regions. The thermal denaturation occurred in the region encompassing the AP2 domain. The difference between the retention time of CBF1 at 4 degrees C and that at 25 degrees C in gel filtration, and the decrease of the sedimentation coefficient, s20,w, caused by the temperature change from 25 to 3 degrees C, strongly suggested that the cold denaturation was accompanied by the extension of the molecule. The possible cold denaturation observed here might be a physiologically important structural response of CBF1 to cold stress.

Glycodelin and β-lactoglobulin, lipocalins with a high structural similarity, differ in ligand binding properties

Human glycodelin, a lipocalin with a high amino acid similarity to β-lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin-A) and seminal plasma (glycodelin-S). We found that the structures of these glycodelins and β-lactoglobulin are similar. Despite this structural similarity, unlike β-lactoglobulin, glycodelin-A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin-A and glycodelin-S is not influenced by the differences in glycosylation or by ligand binding.

Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains: reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy.

The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0, reversible thermal denaturation of TmPGK occurred below 90 degrees C. The corresponding peaks in the partial molar heat capacity function were fitted by a four-state model, describing three well-defined unfolding transitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions and subsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversibly between pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineered C-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2 nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structured intermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiary structure, whereas for C1/2 the intermediate could not be defined with certainty.

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization.

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.

Kinetic study on thermal denaturation of hen egg-white lysozyme involving precipitation

A kinetic study on the thermal denaturation accompanying precipitation of hen egg-white lysozyme was performed at temperatures between 50 and 90°C. Visible precipitation occurred at lysozyme concentrations higher than 10 −5M. Even at the concentration of 10 −6M where no visible precipitation was observed, irreversible and reversible denaturation could be clearly discriminated. The former involves two different reactions with activation energies of approximately 93 and 50 kJ·mol −1. On the other hand, enthalpy and entropy changes in the latter are 443 kJ·mol −1 and 1280 J·K −1·mol −1, respectively, indicating a large conformational change. The contradiction that the denaturation or deactivation reaction fitted first-order reaction kinetics while its rate constant depended on the protein concentration, was resolved by newly proposed schemes. The apparent first-order rate constant obtained experimentally depended on the initial protein concentration being on the order of almost unity. Moreover, it was revealed that the apparent first-order reaction involved a second-order reaction that characterized the aggregation of denatured protein molecules. The theory developed here explained reasonably the thermal denaturation accompanying precipitation that occurs at high protein concentration and at high temperature, and was also successfully applied to the lower concentration range with no accompanying precipitation.

2PA137 T4ファージ分子シャペロン様タンパク質gp57A変性中間体の熱力学的解析

2PA137 T4ファージ分子シャペロン様タンパク質gp57A変性中間体の熱力学的解析

Folding mechanism of three structurally similar beta-sheet proteins.

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly beta-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, deltaGH2O) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences.

Folding mechanism of three structurally similar β‐sheet proteins

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly β-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, ΔG) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences. Proteins 33:107–118, 1998. © 1998 Wiley-Liss, Inc.

312 Effect of diazinon on reversible kinetic parameters of bovine retinal acetyl-Cholinesterase

A kinetic study on the thermal denaturation accompanying precipitation of hen egg-white lysozyme was performed at temperatures between 50 and 90°C. Visible precipitation occurred at lysozyme concentrations higher than 10−5M. Even at the concentration of 10−6M where no visible precipitation was observed, irreversible and reversible denaturation could be clearly discriminated. The former involves two different reactions with activation energies of approximately 93 and 50 kJ·mol−1. On the other hand, enthalpy and entropy changes in the latter are 443 kJ·mol−1 and 1280 J·K−1·mol−1, respectively, indicating a large conformational change. The contradiction that the denaturation or deactivation reaction fitted first-order reaction kinetics while its rate constant depended on the protein concentration, was resolved by newly proposed schemes. The apparent first-order rate constant obtained experimentally depended on the initial protein concentration being on the order of almost unity. Moreover, it was revealed that the apparent first-order reaction involved a second-order reaction that characterized the aggregation of denatured protein molecules. The theory developed here explained reasonably the thermal denaturation accompanying precipitation that occurs at high protein concentration and at high temperature, and was also successfully applied to the lower concentration range with no accompanying precipitation.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0.

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine beta-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using alpha-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of beta-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 microM; kinetic parameters for the substrate hydrolysis were: Km = 32 microM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

Scan-rate dependence in protein calorimetry: the reversible transitions of Bacillus circulans xylanase and a disulfide-bridge mutant.

The stabilities of Bacillus circulans xylanase and a disulfide-bridge-containing mutant (S100C/N148C) were investigated by differential scanning calorimetry (DSC) and thermal inactivation kinetics. The thermal denaturation of both proteins was found to be irreversible, and the apparent transition temperatures showed a considerable dependence upon scanning rate. In the presence of low (nondenaturing) concentrations of urea, calorimetric transitions were observed for both proteins in the second heating cycle, indicating reversible denaturation occurs under those conditions. However, even for these reversible processes, the DSC curves for the wild-type protein showed a scan-rate dependence that was similar to that in the absence of urea. Calorimetric thermograms for the disulfide mutant were significantly less scan-rate dependent in the presence of urea than in the urea-free buffer. The present data show that, just as for irreversible transitions, the apparent transition temperature for the reversible denaturation of proteins can be scan-rate dependent, confirming the prediction of Lepock et al. (Lepock JR, Rithcie KP, Kolios MC, Rodahl AM, Heinz KA, Kruuf J, 1992, Biochemistry 31:12706-12712). The kinetic factors responsible for scan-rate dependence may lead to significant distortions and asymmetry of endotherms, especially at higher scanning rates. This points to the need to check for scan-rate dependence, even in the case of reversible denaturation, before any attempt is made to analyze asymmetric DSC curves by standard thermodynamic procedures. Experiments with the disulfide-bridge-containing mutant indicate that the introduction of the disulfide bond provides additional stabilization of xylanase by changing the rate-limiting step on the thermal denaturation pathway.

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380–386, 1998.

Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea.

Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin (107 kDa) whose cell lytic activity may be preserved for months at -20 degreesC in the presence of 6 M urea, although it decays rapidly in urea-free buffers. This paper describes experiments addressed to unravel the role of urea in HlyA stabilization. Urea up to 8 M inhibits the Ca2+-binding and hemolytic activities of the protein, alters its secondary and tertiary structures, and reduces its tendency to self-aggregation. All these changes are largely reversed upon urea removal by dilution or dialysis, suggesting that they are interrelated. Furthermore, the extent of recovery of the native activities and structural features of alpha-hemolysin that follows urea removal increases with the concentration of urea during the previous phase. Thus, it seems that urea elicits the reversible transition of HlyA to a less active but more stable state whose structure differs significantly from that of the native protein. Moreover dialysis equilibration of the protein with buffers containing 3 M urea induces the formation of a molecular form of HlyA 5-10 times more active than the native protein in the absence of urea. This hyperactive intermediate appears to keep the native secondary structure of HlyA, but with a less compact tertiary structure, that increases the number of exchangeable Ca2+ ions under these conditions. Changes in the intrinsic fluorescence of HlyA also support the notion of a conformational change in the high-activity intermediate. The intermediate is only detected when assayed in the presence of Ca2+ and 3 M urea and can bind a large number of calcium ions (approximately 12 vs approximately 3 for the native protein); it shows a large tendency to self-aggregation and presumably, in the presence of membranes, a similar tendency to irreversible insertion, which may be the reason for its high lytic activity.

Evidence for receptor bound endothelin in renal but not in cardiac tissues from normal rats.

The stoichiometric binding model (Frelin C, Guedin D. Cardiovasc Res 1994;28:1613-1622) implies that most endothelin in tissues is bound onto receptors rather than in a free state. The objective of this study was to assay receptor bound endothelins in normal rat tissues. We first defined acidic conditions that promoted a mild and reversible denaturation of ETA and ETB receptors and that allowed dissociation of bound [125I]endothelin-1. The action of an acid wash on [125I]endothelin-1 binding to cell or tissue homogenates was then investigated. Acid washing of homogenates prepared from rat brain capillary endothelial cells that express prepro endothelin-1 mRNAs unmasked receptor sites. Acid washing of cardiac, lung, brain or liver homogenates did not increase [125I]endothelin-1 binding. An acid wash of kidney homogenates increased 2.2 fold [125I]endothelin-1 binding. Experiments using BQ-123 further indicated that the acid treatment of renal homogenates mainly unmasked ETA receptors. Masked renal ET receptors were mainly localized in the medulla. Treatment of rats with phosphoramidon decreased the density of masked ET receptors in kidney homogenates. As much as 50% of endothelin receptors in renal tissues are masked by endogenous endothelins. Most cardiac receptors are free of bound endothelins. These suggest that endothelins act as local rather than systemic mediators.

Biophysical Characterization of the TraY Protein ofEscherichia coli F Factor

The TraY protein is required for efficient bacterial conjugation by Escherichia coli F factor. TraY has two functional roles: participating in the "relaxosome," a protein-DNA complex that nicks one strand of the F factor plasmid, and up-regulating transcription from the traYI promoter. The traY gene was cloned, and the TraY protein was expressed, purified, and characterized. TraY has a mixed alpha-helix and beta-sheet secondary structure as judged by its circular dichroism spectrum, is monomeric, and undergoes reversible urea denaturation with delta Gu = 6 kcal/mol at 25 degrees C. The kinetics of protein unfolding and refolding, as measured by changes in fluorescence, are complex, suggesting the presence of intermediates or of heterogeneity in the folding reaction. TraY has been classified as a member of the ribbon-helix-helix family of transcription factors but is unusual in appearing to have tandem repeats of the beta alpha alpha motif in the same polypeptide chain. The data presented here show that folding and assembly of the functional (beta alpha alpha)2 unit occurs as an intramolecular reaction and not by cross-folding between different polypeptide chains.

Amine composition influences apparent activity of enzyme in charged film microcapsules

It has been shown that, when captured in charged film microcapsules prepared from spermine alginate, intact viruses retain infectivity, isolated viral proteins retain immunogenicity, and trypsin retains enzymatic activity. However, it was unclear whether the greater anionic strength of hemisulfate residues such as those in carrageenan might alter protein conformation and activity unfavourably in comparison with the lesser influence of alginate carboxylates. Further, the influence of the structure of the amine used to prepare the capsules was largely unknown. To examine these questions, trypsin, used as a model protein, was encased in microcapsules prepared from iota-carrageenan and oligoamines drawn from either the homologous series spermine, spermidine, putrescine or ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepenta-mine. The gross structures of encapsulated and native trypsin were compared by denaturing electrophoresis and their enzymatic activity by the method of Hummel. In all encapsulations SDS PAGE gave no evidence of alteration of protein structure. When encapsulated, the apparent activity of trypsin was reduced by about 60 to 75%, but when the capsules were lysed in hypertonic saline activity was restored. This apparent reduction in activity is attributed to the diffusional barrier imposed by the encapsulating membrane but it should be recognized that it may be the result of reversible denaturation.

Solution Structure of Porcine Delta Sleep-inducing Peptide Immunoreactive Peptide A Homolog of the ShortsightedGene Product

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.