Refolding of urea-denatured tubulin: recovery of nativelike structure and colchicine binding activity from partly unfolded states

Tubulin unfolding in urea proceeds via the formation of a partially unfolded intermediate state, stable in 2 M urea, that unfolds further in higher urea concentrations. The intermediate state had spectroscopic properties reminiscent of a molten globule and negligible colchicine binding activity. Refolding of totally unfolded tubulin in 8 M urea yielded an intermediatelike state characterized by partial burial of tryptophans and partial recovery of secondary and tertiary structures, although colchicine-binding activity of the protein was not regained. Further folding of this intermediatelike state, toward the native conformation, with respect to both structural and functional parameters did not occur. However, a significant percentage of colchicine binding activity and nativelike tertiary structure was recovered when refolding was initiated from partially denatured protein samples, viz., from <1.2 M urea. Thus, although high concentration of urea induced loss of structure and activity was irreversible, the conformational changes induced in restricted regions of tubulin by lower concentrations of urea, which are probably crucial for its various functional properties, could be reversed.     

The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with 'native format' [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.     

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.     

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.     

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.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

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.     

Conformational states bound by the molecular chaperones GroEL and secB: a hidden unfolding (annealing) activity

We have analysed the conformational states of barnase that are bound by the molecular chaperones GroEL and SecB. Line broadening in the NMR spectra of barnase in the presence of chaperone indicates binding of the native state of barnase to both GroEL and SecB, with a dissociation constant of > 3 x 10(-4) M for the GroEL-native barnase complex. GroEL and SecB catalyse the hydrogen-deuterium exchange of amide proteins of barnase that require global unfolding for exchange to occur, indicating that both chaperones bind to a fully unfolded state of barnase. Binding of the denatured state was also detected by a reversible lowering of the melting temperature of barnase in the presence of chaperone. The dissociation constant of the complex between denatured barnase and either chaperone is 5 x 10(-8) M. The chaperone-bound fully unfolded state is a minor conformation that would not be seen by direct observation under physiological conditions, as the folding intermediate of barnase is the most populated state in the complex. The rate-limiting step for exchange of buried amide protons of bound barnase is the unfolding of the folding intermediate, which is retarded > 2000-fold in the complex with GroEL. The reverse refolding step is retarded > 1000-fold by GroEL leading to an EX1 mechanism for exchange. In contrast, unfolding of native barnase is catalysed by > 1000-fold. Thus, molecular chaperones GroEL and SecB have the potential to act in vivo and in vitro as: (1) a folding/transport-scaffold to prevent aggregation of partially folded states by binding; (2) as an annealing-machine to generate continuous unfolding of misfolded states until a low-affinity state is formed; and (3) as an unfoldase to catalyse unfolding of the misfolded states.     

Characterization of a stable intermediate trapped during reversible refolding of Bacillus subtilis alpha-amylase

Bacillus subtilis exocellular alpha-amylase is reversibly refolded after denaturation by guanidine hydrochloride at pH 7 and 37 degrees C. The unfolding-folding transition monitored by intrinsic fluorescence changes and resistance to proteolysis was resolved into a two-state transition. The first step (t1/2 < 1 s) led from D, the totally unfolded state, to C, a stable partially structured state of the protein. This folding intermediate was devoid of any enzyme activity and partially resistant to protease degradation. Calcium was required for the transition from C to N, the native state. This metal did not remain associated with the native form and could be replaced by barium or strontium, but not by magnesium. We discuss the hypothesis that C, the folding intermediate whose further transformation is under kinetic control, is the competent state involved in the secretion process of alpha-amylase.     

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.     

Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate

We investigated the role of a partially folded intermediate that transiently accumulates during lysozyme folding. Previous studies had shown that the partially folded intermediate is located on a slow-folding pathway and that an additional fast direct pathway from the unfolded state to the native state exists. Kinetic double-jump experiments showed that the two folding pathways are not caused by slow equilibration reactions in the unfolded state. Rather, kinetic partitioning occurs very early in lysozyme refolding, giving the molecules the chance to enter the direct pathway or a slow-folding channel. Fitting the guanidinium chloride dependencies of the refolding and unfolding reactions to analytical solutions for different folding scenarios enables us to propose a triangular mechanism as the minimal model for lysozyme folding explaining all observed kinetic reactions: [diagram in text]. All microscopic rate constants and their guanidinium chloride dependencies could be obtained from the experimental data. The results suggest that population of the intermediate during refolding increases the free energy of activation of the folding process. This effect is due to the increased stability of the intermediate state compared to the unfolded state leading to an increase in the free energy of activation (deltaG0) compared to folding in the absence of populated intermediate states. The absolute energy of the transition state is identical on both pathways. The results imply that pre-formed secondary structure in the folding intermediate obstructs formation of the transition state of folding but does not change the nature of the rate-limiting step in the folding process.     

Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization

The equilibrium unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species. Previously, four unfolded species have been detected experimentally. They are Uvf, Uf, UsII, and UsI which have refolding time constants on the millisecond, millisecond to second, second to tens of seconds, and hundreds of seconds time scales, respectively. In the current study, the refolding pathway of the protein was investigated under favorable folding conditions of 0.58 M GdnHCl, pH 5.0, and 15 degrees C. In addition to the above four unfolded species, the presence of a fifth unfolded species was detected. It has a refolding time constant on the order of 2 s under the conditions employed. This new unfolded species is labeled Um, for medium-refolding species. Single-jump refolding experiments monitored by tyrosine burial and by cytidine 2'-monophosphate inhibitor binding indicate that the different unfolded species refold to the native state along independent refolding pathways. The buildup of the different unfolded species upon unfolding of the protein from the native state was monitored by absorbance using double-jump experiments. These experiments were carried out at 15 degrees C and consisted of an unfolding step at 4.2 M GdnHCl and pH 2.0, followed, after a variable delay time, by a refolding step at 0.58 M GdnHCl and pH 5.0. The results of these experiments support the conclusion that the different unfolded species arise from cis-trans isomerizations at the X-Pro peptide bonds of Pro 93, 114, and 117 in the unfolded state of the protein. The rates of these isomerizations were obtained for each of these three X-Pro peptide bonds at 15 degrees C.     

Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.     

Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily

Solvent-induced equilibrium unfolding of a homodimeric class sigma glutathione transferase (GSTS1-1, EC 2.5.1.18) was characterized by tryptophan fluorescence, anisotropy, enzyme activity, 8-anilino-1-naphthalenesulfonate (ANS) binding, and circular dichroism. Urea induces a triphasic unfolding transition with evidence for two well-populated thermodynamically stable intermediate states of GSTS1-1. The first unfolding transition is protein concentration independent and involves a change in the subunit tertiary structure yielding a partially active dimeric intermediate (i.e., N2 left and right arrow I2). This is followed by a protein concentration dependent step in which I2 dissociates into compact inactive monomers (M) displaying enhanced hydrophobicity. The third unfolding transition, which is protein concentration independent, involves the complete unfolding of the monomeric state. Increasing NaCl concentrations destabilize N2 and appear to shift the equilibrium toward I2 whereas the stability of the monomeric intermediate M is enhanced. The binding of substrate or product analogue (i.e., glutathione or S-hexylglutathione) to the protein's active site stabilizes the native dimeric state (N2), causing the first two unfolding transitions to shift toward higher urea concentrations. The stability of M was not affected. The data implicate a region at/near the active site in domain I (most likely alpha-helix 2) as being highly unstable/flexible which undergoes local unfolding, resulting initially in I2 formation followed by a disruption in quaternary structure to a monomeric intermediate. The unfolding/refolding pathway is compared with those observed for other cytosolic GSTs and discussed in light of the different structural features at the subunit interfaces, as well as the evolutionary selection of this GST as a lens crystallin.     

Sequential unfolding of papain in molten globule state

Papain exhibits the characteristics of molten globule under acidic conditions as seen by circular dichroism, fluorescence and ANS binding. Between pH 2.0-2.5 the protein exhibits substantial secondary structure as indicated by far-UV CD spectrum but loses the persistent tertiary interactions of the native state. Enhanced binding of ANS to the state at pH 2.0 in relation to the native and unfolded states at neutral pH indicates a considerable exposure of aromatic side chains. Temperature and guanidine hydrochloride induced unfolding of papain in this state is noncooperative and the transition curves are biphasic in nature. As papain molecule consists of two domains, the results suggest that the domains unfold independently and sequentially.     

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.     

High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA

We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of alpha-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly "softens" at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less "tight". This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, kM, of the protein "native" state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.     

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.     

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.