Differences in the effects of TFE on the folding pathways of human stefins A and B.

Trifluoroethanol (TFE) has been used to probe differences in the stability of the native state and in the folding pathways of the homologous cysteine protein inhibitors, human stefin A and B. After complete unfolding in 4.5 mol/L GuHCl, stefin A refolded in 11% (vol/vol) TFE, 0.75 mol/L GuHCl, at pH 6.0 and 20 degrees C, with almost identical first-order rate constants of 4.1 s-1 and 5.5 s-1 for acquisition of the CD signal at 230 and 280 nm, respectively, rates that were markedly greater than the value of 0.11 s-1 observed by the same two probes when TFE was absent. The acceleration of the rates of refolding, monitored by tyrosine fluorescence, was maximal at 10% (vol/vol) TFE. Similar rates of refolding (6.2s-1 and 7.2 s-1 for ellipticity at 230 and 280 nm, respectively) were observed for stefin A denatured in 66% (vol/vol) TFE, pH 3.3, when refolding to the same final conditions. After complete unfolding in 3.45 mol/L GuHCl, stefin B refolded in 7% (vol/vol) TFE, 0.57 mol/L GuHCl, at pH 6.0 and 20 degrees C, with a rate constant for the change in ellipticity at 280 nm of 32.8 s-1; this rate was only twice that observed when TFE was absent. As a major point of distinction from stefin A, the refolding of stefin B in the presence of TFE showed an overshoot in the ellipticity at 230 nm to a value 10% greater than that in the native protein; this signal relaxed slowly (0.01 s-1) to the final native value, with little concomitant change in the near-ultraviolet CD signal; the majority of this changes in two faster phases. After denaturation in 42% (vol/vol) TFE, pH 3.3, the kinetics of refolding to the same final conditions exhibited the same rate-limiting step (0.01 s-1) but were faster initially. The results show that similarly to stefin A, stefin B forms its hydrophobic core and predominant part of the tertiary structure faster in the presence of TFE. The results imply that the alpha-helical intermediate of stefin B is highly structured. Proteins 1999;36:205-216.     

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.     

Fast folding of the HIV-1 and SIV gp41 six-helix bundles

Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-Trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more α-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivation indicated that the active site of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

Effect of trifluoroethanol on the conformational stability of a hyperthermophilic esterase: a CD study

The conformational stability of the hyperthermophilic esterase AFEST from Archeoglobus fulgidus against the denaturing action of 2,2,2-trifluoroethanol (TFE) has been investigated by means of circular dichroism (CD) measurements. At room temperature far-UV and near-UV CD spectra point out the occurrence of a co-operative transition from the native structure to a denatured state characterized by a high content of alpha-helix. The TFE concentration at half-completion of the transition proves to be 3.5 M (25% v v(-1)), by recording the molar ellipticity at both 222 and 276 nm. Thermal transition curves of AFEST in the absence and in the presence of TFE indicate a significant stability decrease on increasing the TFE concentration. The denaturation temperature is 99 degrees C for native AFEST, but becomes 85 degrees C at 1.4 M TFE (10% v v(-1)), and 56 degrees C at 2.8 M TFE (20% v v(-1)). It is also shown that, even though AFEST is very resistant to temperature, its resistance towards the denaturing action of TFE is similar to that of mesophilic proteins, including an esterase from Escherichia coli, AES. The proposal of a general mechanism for the TFE action on globular proteins leads to a reliable rationale of experimental data.     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of folding and unfolding reactions of cytochrome b5

The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b(5) containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (DeltaG(uw)) of 26.5 kJ mol(-1). This stability is much higher than that reported for the corresponding form of the apoprotein (approximately 7 kJ mol(-1)). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying approximately 30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (k(uw)) are 4.5 x 10(-5) s(-1) and 5.2 x 10(-6) s(-1) at 25 degrees C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis-trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b(5), the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme-protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol(-1) exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln k(obs) versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a "fast track" leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.     

Conformational changes and inactivation of calf intestinal alkaline phosphatase in trifluoroethanol solutions

The changes in activity and unfolding of calf intestinal alkaline phosphatase (CIP) during denaturation in different concentrations of trifluoroethanol (TFE) have been investigated by far-ultraviolet circular dichroism and fluorescence emission spectra. Unfolding and activation rate constants were measured and compared, the activation and inactivation courses were much faster than that of unfolding, which suggests that the active site of CIP containing two zinc ions and one magnesium ion is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole. However, compared to other metalloenzymes, CIP is inactivated at higher concentrations of TFE as denaturant.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Conformational transitions of thioredoxin in guanidine hydrochloride

Spectral and hydrodynamic measurements of thioredoxin from Escherichia coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25 degrees C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 +/- 0.2 s. Refolding measurements in 2 M denaturant reveal three kinetic phases having relaxation times of 0.54 +/- 0.23, 14 +/- 6, and 500 +/- 130 s, accounting for 12 +/- 2%, 10 +/- 1%, and 78 +/- 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatured state have an activation enthalpy of 22 +/- 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerization of proline-76 to its cis isomer prior to refolding.     

Ca(II)- and Tb(III)-induced stabilization and refolding of anticoagulation factor I from the venom of Agkistrodon acutus

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF I, holo-ACF I, and Tb(3+)-reconstituted ACF I in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism. Metal ions were found to increase the structural stability of ACF I against GdnHCl and thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF I and Tb(3+)-ACF I is a two-state process with no detectable intermediate state(s), whereas the GdnHCl-induced unfolding/refolding of holo-ACF I in the presence of 1 mM Ca(2+) follows a three-step transition, with intermediate state a (Ia) and intermediate state b (Ib). Ca(2+) ions play an important role in the stabilization of the Ia and Ib states. The decalcification of holo-ACF I shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, whereas the reconstitution of apo-ACF I with Tb(3+) ions shifts the initial zone of denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.0 M GdnHCl) at which refolding from the fully denatured state of apo-ACF I to the Ib state of holo-ACF I or to the native state of Tb(3+)-ACF I can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF I, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ions-induced refolding provide evidence that the compact Tb(3+)-binding region forms first, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

根霉葡萄糖淀粉酶的复性复活动力学研究

本文利用荧光、紫外差光谱研究了根霉葡萄糖淀粉酶在盐酸胍变性后的复性、复活动力学。结果表明,该酶在小于4mol/L盐酸胍中变性是可逆的,其复性过程遵循一级反应方程。酶复活过程是由两个一级反应组成的复合反应,构象变化速度与复活过程中较快的反应速度相差无几,这可能是在Trp及Tyr微区的构象变化基本完成之后,酶活力恢复还没有完成造成的。     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation.

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.