GdnHCl Concentration Research Articles

Operation conditions of enzyme refolding by chaperonin and recycle system using ultrafiltration

Abstract To clarify the efficient refolding conditions of enzymes using chaperonin GroE from Escherichia coli , the effects of various factors on the chaperonin-mediated refolding of enzymes from 4M guanidine hydrochloride (GdnHCl) were investigated. Three enzymes, Bacillus subtilis α-amylase, bovine deoxyribonuclease I (DNase I) and yeast enolase were used as the model systems. In all enzymes, the maximum recovery of activities (90–150% with respect to the enzyme activities before denaturation) was attained in the presence of 2mM ATP and 2–5-fold molar excess of GroE 21-mer or GroEL 14-mer over enzyme molecules. Since the recovery of enzyme activity by GroEL is close to that by GroE, GroEL as well as GroE are applicable for the refolding systems of these enzymes. GroE significantly enhanced the recovery of enzyme activities at around 25–40 °C, pH 6–9 and up to rather high final GdnHCl and enzyme concentrations. Therefore, the chaperonin efficiently mediates the enzyme refolding under wide operation conditions. To test the reusability of chaperonins in enzyme refolding, GroEL was separated from refolded enzymes by ultrafiltration and recycled. GroEL repeatedly mediated the refolding of enzymes by choosing appropriate membranes. Therefore, protein refolding processes based on chaperonins are promising.

Scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies

Denaturation studies with guanidine HCl (GdnHCl) were performed to test the relationship between scrapie infectivity and properties of scrapie-associated prion protein (PrP(Sc)). Large GdnHCl-induced reductions in infectivity were associated with the irreversible elimination of both the proteinase K resistance and apparent self-propagating converting activity of PrP(Sc). In intermediate GdnHCl concentrations that stimulate converting activity and partially disaggregate PrP(Sc), both scrapie infectivity and converting activity were associated with residual partially protease-resistant multimers of PrP(Sc).

A Comparison of the Denaturants Urea and Guanidine Hydrochloride on Protein Refolding

The effect of the denaturants guanidine hydrochloride (GdnHCl) and urea on enzyme activity has been investigated. Inactivation of hen egg white lysozyme and bovine carbonic anhydrase was monitored by enzyme activity assays, and fluorescence spectroscopy was used to detect conformational changes. The results show that lysozyme and carbonic anhydrase were inactivated at GdnHCl and urea concentrations which were much lower than those required to cause denaturation. Ionic strength effects, examined by addition of NaCl, were found to significantly reduce the activity of lysozyme and carbonic anhydrase to different extents. Denaturant type was also found to affect refolding yields. The refolding of 0.015mgml-1 reduced, denatured lysozyme resulted in a maximum 65% recovery of activity from GdnHCl and 50% from urea. The first order rate constants for refolding were found to be 2.4×10-3 s-1 and 2.5×10-3 s-1 for GdnHCl and urea denatured lysozyme respectively. It is believed that the differences in yield from these two denaturants are due to their effect on protein aggregation. Concentrations of GdnHCl greater than 0.01M present during refolding greatly reduced the yield of active protein.

Disruption of the disulfide bonds of recombinant murine interleukin-6 induces formation of a partially unfolded state.

A chemical modification approach was used to investigate the role of the two disulfide bonds of recombinant murine interleukin-6 (mIL-6) in terms of biological activity and conformational stability. Disruption of the disulfide bonds of mIL-6 by treatment with iodoacetic acid (IAA-IL-6) or iodoacetamide (IAM-IL-6) reduced the biological activity, in the murine hybridoma growth factor assay, by 500- and 200-fold, respectively. Both alkylated derivatives as well as the fully reduced (but not modified) molecule (DTT-IL-6) retained a high degree of alpha-helical structure as measured by far-UV CD (37-51%) when compared to the mIL-6 (59%). However, the intensity of the near-UV CD signal of the S-alkylated derivatives was very low relative to that of mIL-6, suggesting a reduction in fixed tertiary interactions. Both IAA-IL-6 and IAM-IL-6 exhibit native-like unfolding properties at pH 4.0, characteristic of a two-state unfolding mechanism, and are destabilized relative to mIL-6, by 0.3 +/- 1.6 and 2.4 +/- 1.2 kcal/mol, respectively. At pH 7.4, however, both modified proteins display stable unfolding intermediates. These intermediates are stable over a wide range of GdnHCl concentrations (0.5-2 M) and are characterized by increased fluorescence quantum yield and a blue shift of lambda(max) from 345 nm, for wild-type recombinant mIL-6, to 335 nm. These properties were identical to those observed for DTT-IL-6 in the absence of denaturant. DTT-IL-6 appears to form a partially unfolded and highly aggregated conformation under all conditions studied, as showed by a high propensity to self-associate (demonstrated using a biosensor employing surface plasmon resonance), and an increased ability to bind the hydrophobic probe 8-anilino-1-naphthalenesulfonic acid. The observed protein concentration dependence of the fluorescence characteristics of these mIL-6 derivatives is consistent with the aggregation of partially folded forms of DTT-IL-6, IAM-IL-6, and IAA-IL-6 during denaturant-induced unfolding. For all forms of the protein studied here, the aggregated intermediates unfold at similar denaturant concentrations (2.1-2.9 M GdnHCl), suggesting that the alpha-helical structure and nonspecific hydrophobic interprotein interactions are of similar strength in all cases.

Thermodynamic stability of annexin V E17G: equilibrium parameters from an irreversible unfolding reaction.

Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t1/2 = 54.7 degrees C (heating rate of 2.34 K min-1), delta H0 = 690 kJ mol-1, and delta Cp = 10.3 kJ mol-1 K-1 which correspondends to a value of delta G0D (20 degrees C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration C1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the delta Gapp versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature delta t1/2 (= 18 degrees C) when the GdnHCl concentration is increased from 0 to 1 M. "Regular" proteins experience an average decrease in the transition temperature of 8 +/- 2 degrees C per 1 M change in the concentration of GdnHCl.

Unfolding and aggregation‐associated changes in the secondary structure of D‐glyceraldehyde‐3‐phosphate dehydrogenase during denaturation by guanidine hydrochloride as monitored by FTIR

The secondary structure of native D-glyceraldehyde-3-phosphate dehydrogenase was compared with its partially folded intermediate and aggregated states obtained during guanidine hydrochloride (GdnHCl) denaturation using transmission Fourier transform infrared (FTIR) and micro-FTIR measurements. The changes in the secondary structures indicated a partially folded intermediate formed in 0.1M GdnHCl solution without visible aggregation. Increasing the GdnHCl concentration resulted in aggregation of the enzyme along with changes in the secondary structure. Although similar relative amounts of the secondary structure were found in the aggregated and native states of the enzyme, the temporal variation of the secondary structure revealed a difference in the β-sheet structure between the aggregated and native states, suggesting that the aggregation resulted from further unfolding of the enzyme. In addition, FTIR data suggest that such aggregates are most likely mediated by specific intermolecular interactions and that the predominant driving force involved in aggregation may be a hydrophobic interaction between exposed surfaces of partially folded intermediates. © 1997 John Wiley & Sons, Inc. Biospect 3: 121–129, 1997

Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase.

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.

Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3

The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1–0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of ≤ 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome- c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the ‘denatured’ P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80–90% lost from 0.5–-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.

RhNGF slow unfolding is not due to proline isomerization: Possibility of a cystine knot loop‐threading mechanism

The unfolding of recombinant human beta-NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 degrees C. Fluorescence spectroscopy, size-exclusion and reversed-phase HPLC, ultra-centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little beta-sheet structure, but retained residual structure in the tryptophan indole and high-field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half-life of 23 h at 25 degrees C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N-terminus. A model for the slow unfolding reaction is proposed where the N-terminus threads through the cystine knot to form M2, a loop-threading reaction, increasing the conformational freedom of the denatured state.

Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5′-phosphate

The α-amino group of Lys-1 and the ϵ-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5′-phosphate (PLP) separately. The effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decreased obviously during denaturation by low concentrations of GdnHCl indicating conformational change of the active site is a parallel event to the inactivation of the enzyme. Time-correlated fluorescence lifetime measurements revealed the existence of two conformational states of PLP-modified RNase A and a shift of the conformation in favor of the slow-decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole.

Stabilization of barstar by chemical modification of the buried cysteines.

The internal packing of residues in the small monomeric protein barstar was severely perturbed by chemical modification of the two buried cysteine residues with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) after prior unfolding of the protein using guanidine hydrochloride (GdnHCl). The modification produces mixed disulfides between 5-thio(2-nitrobenzoic acid) and the two Cys residues. To understand the effects of the modification of the individual cysteine residues, Cys40 and Cys82, the modification was also carried out on the two single Cys --> Ala mutant forms of barstar, C40A and C82A, whose structures, activities, and stabilities were first shown to be similar to those of wt barstar. Equilibrium GdnHCl-induced denaturation studies on wt barstar show that the modification causes the midpoint of the denaturation curve to increase by 0.6 M and the stability to increase by 1.3 kcal mol-1. Both C40A and C82A also denature at higher concentrations of GdnHCl after modification. Modification of Cys40 has approximately the same stabilizing contribution as does modification of Cys82. The structures of the modified and unmodified proteins have been compared using circular dichroism (CD) spectroscopy, UV difference absorption spectroscopy, and fluorescence spectroscopy. It is shown that the 5-thio(2-nitrobenzoic acid) groups introduced by reaction with DTNB are buried in hydrophobic environments in the modified C40A and C82A mutant proteins, as well as in modified wt barstar. The far-UV CD spectra of the modified and unmodified proteins are similar, but the mean residue ellipticity at 220 nm of wt barstar is reduced by 30% upon modification. Such a decrease is not seen for either C40A or C82A. The barnase-inhibiting activities of the three modified proteins are shown to be similar to those of the corresponding unmodified proteins. Thus, the severe perturbations of the internal packing, which result in a significant increase in stability, do not appear to affect the overall fold of barstar.

Kinetic and thermodynamic studies of the folding/unfolding of a tryptophan-containing mutant of ribonuclease A.

Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85% of the activity of the wild-type protein. Single-jump unfolding experiments showed that the unfolding pathway of the Trp mutant contains a fast and a slow phase similar to those seen previously for the wild-type protein, indicating that the mutation did not alter the unfolding pathway significantly. The activation energy of the slow-unfolding phase suggested that proline isomerization is involved, with the Trp residue presumably reporting on changes in its local environment. Single-jump refolding experiments revealed the presence of GdnHCl-independent burst phase and a native-like intermediate, most likely IN, on the folding pathway. Single-jump refolding data at various final GdnHCl concentrations were fit to a kinetic folding model involving two pathways to the native state; one pathway involves the intermediate IN, and the other is a direct one to the native state. This model provides site-specific information, since Trp92 monitors the formation of local structure only in the neighborhood of that residue. Double-jump refolding experiments permitted the detection of a previously reported, hydrophobically collapsed intermediate, I phi. The refolding data support the hypothesis that the region around position 92 is a chain-folding initiation site in the folding pathway.

Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates

The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25°C has been characterized using rapid-mixing techniques. Ear;lier studies had established the validity of the three-state US⇌UF⇌N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where USand UFare the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1and IS2as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1on folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, INis not fully populated on the US→IS1→IN→N pathway because the rate of its formation is so slow that the US⇌UF⇌N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US→IS1→INtransition is so fast that INis fully populated. In 0.6 M GdnHCl, INappears not to be fully populated because an alternative folding pathway, US→IS2→N, becomes available for the folding of US, in addition to the US→IS1→INN pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1and IM2, that form within 2 ms on the US→IM1→IS1→IN→N and US→IM2→IS2→N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

Denaturing Behavior of Glutathione Reductase from Cyanobacterium Spirulina maxima in Guanidine Hydrochloride

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low Gdn-HCl concentrations ( C m = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturirng agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly! suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16, 115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl ( C m = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.

Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride.

Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curves obtained at 14 different temperatures in the range 273-323 K have been used in conjunction with thermally-induced denaturation curves obtained in the presence of 15 different concentrations of GdnHCl to characterize the thermodynamics of cold and heat denaturation of barstar. The linear free energy model has been used to determine the excess changes in free energy, enthalpy, entropy, and heat capacity that occur on denaturation. The stability of barstar in water decreases as the temperature is decreased from 300 to 273 K. This decrease in stability is not accompanied by a change in structure as monitored by measurement of the mean residue ellipticities at both 222 and 275 nm. When GdnHCl is present at concentrations between 1.2 and 2.0 M, the decrease in stability with decrease in temperature is however so large that the protein undergoes cold denaturation. The structural transition accompanying the cold denaturation process has been monitored by measuring the mean residue ellipticity at 222 nm. The temperature dependence of the change in free energy, obtained in the presence of 10 different concentrations of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K. Values of the thermodynamic parameters governing the cold and the heart denaturation of barstar have been obtained with high precision by analysis of these bell-shaped stability curves. The change in heat capacity accompanying the unfolding reaction, delta Cp, has a value of 1460 +/- 70 cal mol-1 K-1 in water. The dependencies of the changes in enthalpy, entropy, free energy, and heat capacity on GdnHCl concentration have been analyzed on the basis of the linear free energy model. The changes in enthalpy (delta Hi) and entropy (delta Si), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the folded state, both have a negative value at low temperatures. With an increase in temperature delta Hi makes a less favorable contribution, while delta Si makes a more favorable contribution to the change in free energy (delta Gi) due to this interaction. The change in heat capacity (delta CPi) that occurs on preferential interaction of GdnHCl with the unfolded form has a value of only 53 +/- 36 cal mol-1 K-1 M-1. The data validate the linear free energy model that is commonly used to analyze protein stability.

Amphiphilic α-helical structure in water stabilized by dioctadecyl chain

The peptides H-(Leu-Aib-Lys-Aib-Aib-Lys-Aib)3-X [X = OMe, P21OMe; X =-Ala-N(C18H37)2, P21D], have been synthesized as models for biologically active peptides having long alkyl chains, and the effect of the alkyl chain on the peptide conformation has been studied. CD measurements revealed that the α-helix content of P21D in water was higher than in P21OMe, while their conformations were nearly the same in methanol. Guanidine hydrochloride (GdnHCl), which is a denaturant of proteins, increased the helix content of P21D, but decreased the helix content of P21OMe. The stabilization of the helical structure of P21D at high concentrations of GdnHCl is ascribed to a reduction in the electrostatic repulsion between the ammonium groups of P21D at high ionic strength. It is notable that in water the major fraction of P21D exists as a monomer, but the number of P21D molecules involved in higher aggregates is significant, as revealed by dynamic light scattering measurement. A short-chain peptide, H-Leu-Aib-Lys-Aib-Aib-Lys-Aib-AlaN(C18H37)2, formed much larger aggregates in water than P21D. It probably formed micelles without taking on a helical conformation. Therefore, the connection of two long alkyl chains at the C terminal of a longchain amphiphilic helical peptide is a suitable method for stabilizing the helix conformation without being accompanied by severe aggregation.

Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone

The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)

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The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25°C has been characterized using rapid-mixing techniques. Ear;lier studies had established the validity of the three-state US⇌UF⇌N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where USand UFare the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1and IS2as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1on folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, INis not fully populated on the US→IS1→IN→N pathway because the rate of its formation is so slow that the US⇌UF⇌N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US→IS1→INtransition is so fast that INis fully populated. In 0.6 M GdnHCl, INappears not to be fully populated because an alternative folding pathway, US→IS2→N, becomes available for the folding of US, in addition to the US→IS1→INN pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1and IM2, that form within 2 ms on the US→IM1→IS1→IN→N and US→IM2→IS2→N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

Evidence for an equilibrium intermediate in the folding-unfolding pathway of a transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein.

TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys-->Ala mutant (PE40 delta Cys) of a 40,000-dalton segment (PE40) of Pseudomonas exotoxin (PE). The guanidine hydrochloride (Gdn-HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichroism (CD), and high-performance size exclusion chromatography (HPSEC). The equilibrium unfolding of both proteins involves at least one intermediate (I). In the I state(s), which may be induced by 1.3-2.0 M Gdn-HCl, the tertiary structure is fully or partially collapsed as detected by tryptophan fluorescence and near-UV CD, but the protein largely retains the native secondary structure and a semicompact shape as judged by far-UV CD and HPSEC, respectively. Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermediate (but not at higher) Gdn-HCl concentrations, suggesting that self-association is possibly mediated by thermodynamically stable, partially unfolded I states. The kinetics of refolding of TP40 upon dilution of Gdn-HCl involve two or more phases. Re-formation of secondary structure occurs rapidly (t 1/2 < 10 s) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence. The midpoint (Tm) of the thermal unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD. These data suggest that Gdn-HCl and temperature can induce conformation(s) of TP40 that are distinct from native (N) and unfolded (U) states.(ABSTRACT TRUNCATED AT 250 WORDS)