Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies

Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.     

The actigraph data analysis software: I. A novel approach to scoring and interpreting sleep-wake activity

Intersubunit cross-linked creatine kinase (CK) has been prepared with the cross-linking reagent dithiobis(succinimidyl propionate) (DTSP). Unfolding of cross-linked CK during denaturation by guanidine hydrochloride (GuHCl), as monitored by intrinsic fluorescence, circular dichroism and fluorescence of the hydrophobic probe, 1-aniline-naphthalene-8-sulfonate (ANS), occurs in two stages with increasing GuHCl concentration. The process is similar to that of the unmodified enzyme, but in the second stage, conformational changes of the cross-linked enzyme need higher concentration of GuHCl, suggesting that there is a stable intermediate during its unfolding transition and the intermediate is stabilized by intersubunit cross-linkage.     

Unfolding and refolding of the N-terminal fragments of staphylococcal nuclease R in guanidine hydrochloride

The conformational and activity changes of a family of peptide fragments of staphylococcal nuclease R, which extend from residues -6 to 102, -6 to 110, -6 to 121, -6 to 135, and -6 to 141, during unfolding and refolding in different concentrations of guanidine hydrochloride have been studied. The studies indicate that the conformational stability in guanidine hydrochloride solution of the N-terminal fragment increases with increasing chain length, and that interaction and recognition between amino acid residues which are related to formation of the native conformation also increase with growth of the peptide chain, but such interaction becomes effective only when the polypeptide chain reaches a certain length. The changes in conformation and catalytic activity of the N-terminal fragments during unfolding and refolding demonstrate that conformational adjustments are necessary during chain elongation to generate the native conformation of a biologically active protein.     

Cold denaturation of ubiquitin

Temperature induced unfolding of bovine ubiquitin in solutions with different concentrations of guanidinium hydrochloride (GdmCl) has been measured using differential scanning calorimetry. It has been shown that at high concentrations of GdmCl the ubiquitin molecule can undergo both heat and cold induced denaturation. Analysis of the enthalpy of unfolding of ubiquitin in the presence of GdmCl shows a good agreement with the thermodynamic denaturant binding model. The unfolding Gibbs energy is found to change linearly with guanidine concentration up to zero denaturant concentration.     

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.     

A 19F-NMR study of the equilibrium unfolding of membrane-associated D-lactate dehydrogenase of Escherichia coli

Partially folded protein intermediates have been observed by 19F-NMR spectroscopy during the equilibrium unfolding of the membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli by a denaturant, guanidine hydrochloride (Gdn.HCl). The results from 19F-NMR and circular dichroism spectroscopic studies suggest that the intermediates observed at low Gdn.HCl concentrations (< 3.5 M) exhibit features similar to "molten globules" that contain considerable amounts of secondary and tertiary structure. The results of 19F-NMR studies on 5F-Trp-labeled D-LDH, such as the chemical shift changes, nuclear Overhauser effect, and solvent-induced isotopic shift effect, show that different regions of D-LDH unfold nonuniformly in Gdn.HCl in the presence of lysophosphatidylcholine. The polypeptide appears to unfold in a general order from the carboxyl end to the amino end, in agreement with previous findings from our laboratory that the carboxyl-terminal region of D-LDH is largely exposed to the solvent while the amino-terminal region is buried in the protein core. The structure of the partially unfolded intermediate forms of D-LDH is stabilized in the presence of lipid-like detergents, such as lysophosphatidylcholine.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state

The unfolding kinetics of horse cytochrome c in the oxidized state has been studied at 10, 22, and 34 degreesC as a function of guanidine hydrochloride (GdnHCl) concentration. Rapid (millisecond) measurements of far-UV circular dichroism (CD) as well as fluorescence quenching due to tryptophan to heme excitation energy transfer have been used to monitor the unfolding process. At 10 degreesC, the decrease in far-UV CD signal that accompanies unfolding occurs in two phases. The unobservable burst phase is complete within 4 ms, while the slower phase occurs over tens to hundreds of milliseconds. The burst phase unfolding amplitude increases cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degreesC. In contrast, no burst phase change in fluorescence occurs during unfolding at 10 degreesC. At 22 and 34 degreesC, both the fluorescence-monitored unfolding kinetics and the far-UV CD-monitored unfolding kinetics are biphasic. At both temperatures, the two probes yield burst phase unfolding transitions that are noncoincident with respect to the transition midpoints as well as the dependency of the burst phase amplitudes on GdnHCl concentration. The results suggest that at least two kinetic unfolding intermediates accumulate during unfolding. One burst phase intermediate, IU1, has lost virtually all the native-state secondary structure, while the other burst phase intermediate, IU2, has lost both secondary structure and native-like compactness. The presence of kinetic unfolding intermediates is also indicated by the nonlinear dependence of the logarithm of the apparent unfolding rate constant on GdnHCl concentration, which is particularly pronounced at 10 and 22 degreesC. Analysis of the burst phase unfolding transitions obtained using the two probes shows that the stabilities of IU1 and IU2 decrease steadily with an increase in temperature from 10 to 34 degreesC, suggesting that the structures present in them are stabilized principally by hydrogen bonding interactions.     

Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochloride and a non-ionic denaturant, urea

The effects of non-ionic and ionic denaturation and denaturation/renaturation on the native structure of alpha-crystallin at room temperature were examined. Native alpha-crystallin, at concentrations above and below the previously reported critical micelle concentration (CMC) range, was denatured by varying concentrations of urea and guanidine hydrochloride. The resulting denatured samples were examined by gel filtration fast performance liquid chromatography (FPLC), circular dichroism spectropolarimetry (CD), and transmission electron microscopy. Elution peak samples from gel filtration chromatography with sufficiently high concentrations were examined for subunit composition by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The studies presented herein demonstrate that the denaturation and renaturation of alpha-crystallin via non-ionic urea denaturation results in different renaturation species, depending upon the initial concentration of alpha-crystallin which is denatured and the concentration of urea, including certain species which, by gel filtration FPLC, have an apparent molecular weight greater than the native 800 kD aggregate. Transmission electron microscopy has also demonstrated the existence of a high molecular weight aggregate form for denatured samples. Ionic dissociation, in contrast, proceeds much in the same manner above and below the CMC range, the major difference occurring at 2 M guanidine hydrochloride. alpha B-crystallin is preferentially removed from the native alpha-crystallin aggregate upon treatment with 2 M guanidine hydrochloride indicating, once again, differences between the two subunits. Above and below the CMC range, dissociation with guanidine hydrochloride appears to plateau after 4 M guanidine hydrochloride as indicated by the presence of two apparent homotetrameric species and no further dissociation of these species with increasing guanidine hydrochloride concentrations. CD demonstrates that some secondary structure, which is lost with lower concentrations of alpha-crystallin, is still present when concentrations of alpha-crystallin, well above the critical micelle concentration range, are treated with high concentrations of urea at room temperature. In contrast, concentrations both above and below the CMC range demonstrate a significant loss of secondary structure upon treatment with 2 M guanidine hydrochloride. Finally, ionic denaturation and subsequent renaturation results in the formation of a species which is functionally incapable of protecting gamma-crystallin from heat-induced aggregation.     

Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule

The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods. Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5. The transition between the A form and the N state is completely reversible. UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations. The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation. Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water. Surprisingly, high concentrations of denaturant are required to unfold the A form. For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7. The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding. Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.     

Successful coping with declining objective life style by institutionalized long-term patients. Results of a follow-up analysis of quality of life in long-term care institutions with the Zurich Quality of Life Inventory

Folding of cytochrome c from its low pH guanidine hydrochloride (Gdn-HCl) denatured state revealed a new intermediate, a five-coordinate high spin species with a water molecule coordinated to the heme. Incorporation of this five-coordinated intermediate into the previously reported ligand exchange model can quantitatively account for the observed folding kinetics. In this new model, unfolded cytochrome c is converted to its native structure through an obligatory folding intermediate, the histidine-water coordination state, whereas the five-coordinate state and a bis-histidine state are off-pathway intermediates. When the concentration of Gdn-HCl in the refolding solution was increased, an acceleration of the conversion from the bis-histidine coordinated state to the histidine-water coordinated state was observed, demonstrating that the reaction requires unfolding of the mis-organized polypeptide structure associated with the bis-histidine state.     

Charge-shielding and the "paradoxical" stimulation of tubulin polymerization by guanidine hydrochloride

Low concentrations of guanidine hydrochloride (GuHCl) increase the rate (and to a lesser degree, the extent) of tubulin polymerization as assessed by light scattering. Maximum enhancement occurs at 120-160 mM GuHCl followed by decreases at higher GuHCl. The latent period is decreased, and there is a 3-4 fold reduction in the critical concentration of polymerization. Electronmicrographs reveal microtubules in the controls and an increasing fraction of total polymers present as aberrant microtubules as the GuHCl concentration is increased from 20 to 100 mM. The GuHCl effect is markedly reduced, but not abolished, in tubulin S (in which the anionic C termini of both monomers have been removed). The GuHCl-induced polymerization has an absolute requirement for GTP and taxol or DMSO, is very sensitive to podophyllotoxin inhibition, and can overcome urea-mediated inhibition of polymerization. Guanidinium analogues mimic the GuHCl effect roughly as a function of the number of potential hydrogen bonds. The anions of the guanidine salts superimpose their inhibitory action on the guanidinium cation effect according to the lyotropic series. At higher GuHCl concentrations (peak effect 500-700 mM), a different polymer (type II) is formed that is GTP and taxol independent, but whose polymerization is retarded but not prevented by podophyllotoxin. Its structure resembles the fibrillar network seen in unfolding intermediates of other proteins. We conclude that both charge and hydrogen-bonding ability are major contributors to the GuHCl-induced promotion of tubulin polymerization, and that charge-shielding is likely to be the basis for this effect.     

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.     

The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure

It is generally considered that intermediates of protein folding contain partially formed native-like secondary structures. In contrast, we recently reported that the kinetic folding intermediate of bovine beta-lactoglobulin contains non-native alpha-helical structures. To understand the mechanism that stabilizes the non-native intermediate, we characterized by circular dichroism (CD) the equilibrium unfolding transition of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl) at pH 2 and 4 degrees C. The unfolding transition measured by near-UV CD preceded the transition measured by far-UV CD, indicating the accumulation of the intermediate state. The far-UV CD spectrum of the intermediate, obtained by global fitting analysis of the CD spectra in the presence of various concentrations of Gdn-HCl, was similar to the burst-phase intermediate observed in the refolding kinetics, and contained non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-trifluoroethanol (TFE) increased the helical content of the equilibrium intermediate, although the protein still assumed the native structure in the absence of Gdn-HCl. A phase diagram of the conformational states, i.e. the alpha-helical intermediate, unfolded and native states, against the concentration of TFE and Gdn-HCl was constructed. This indicated that, because of the high helical preference of the amino acid sequence of beta-lactoglobulin, the helical region protrudes into the boundary between the native and unfolded states, resulting in non-monotonic accumulation of the helical intermediate upon equilibrium unfolding of the native beta-sheet structure. This is the first observation to indicate that a non-native alpha-helical intermediate accumulates during equilibrium unfolding of a predominantly beta-sheet protein.     

The folding kinetics and thermodynamics of the Fyn-SH3 domain

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.     

The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells

The guanidine hydrochloride (GdnHCl)- and urea-induced equilibrium denaturation of recombinant polyomavirus (Py) major capsid protein VP1 was studied by circular dichroism and fluorescence spectroscopy. Both secondary and tertiary structures of PyVP1 were shown to be disrupted in the presence of denaturants. Although the far-UV circular dichroism (CD) spectra of PyVP1 in the denaturants exhibit similar two-phase transition as those obtained from the fluorescence measurements, the unfolding of PyVP1 in GdnHCl was shown to be more complex than a similar two-state mechanism. The presence of unfolding intermediates is manifested by the noncoincidence of transitions when detected by different probes. The unfolding intermediate appeared to be stabilized by 1 M NaCl. Addition of Ca2+ and 2-mercaptoethanol does not show significant effect on the conformational stability of PyVP1. Unfolding of PyVP1 in GdnHCl was shown to be an irreversible process.     

Effect of pH on formation of a nativelike intermediate on the unfolding pathway of a Lys 73 --> His variant of yeast iso-1-cytochrome c

Previous work on a Lys 73 --> His (H73) variant of iso-1-cytochrome c at pH 7.5 [Godbole et al. (1997) Biochemistry 36, 119-126] showed that this variant unfolds through a nativelike intermediate that has properties consistent with replacement of the Met 80 heme ligand by His 73. Here, the pH dependence of the equilibrium unfolding of the wild type (WT) and H73 proteins have been investigated, since a characteristic pH dependence is expected for the stability of an intermediate stabilized by histidine-heme ligation. Stability has been evaluated using guanidine hydrochloride and pH denaturation methods. Above pH 5, the m-values from guanidine hydrochloride denaturation of the WT and H73 variants remain significantly different, consistent with continued population of this intermediate. At pH 4.5 the m-values for the two proteins are within error the same. To assess stability at lower pH, acid denaturation was carried out. The midpoint is about 3.3 for both proteins but the transition is broader for the H73 protein, suggestive of intermediates again being populated during the unfolding of the H73 protein at this lower pH. Heme ligation by Met 80 was monitored (695 nm absorbance) during gdnHCl (pH 4.5 and 5.0) and acid denaturation, confirming, respectively, the absence and presence of intermediates. A thermodynamic analysis demonstrates that this complex pH dependence for the presence of histidine ligation induced intermediates is expected and implicates a titratable group with a pKa of approximately 6.6. The analysis also demonstrates when the pH dependences of global stability and stability of an intermediate differ significantly, population of folding intermediates as a function of pH will show novel behavior.     

Protein denaturation: a small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c

Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (Rg) shows a cooperative transition with increasing denaturant with a similar Cm to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.     

Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride

The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).     

Analysing functional connectivity in brain slices by a combination of infrared video microscopy, flash photolysis of caged compounds and scanning methods

The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of alpha-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.