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1 Division of Physical Chemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, UK.
Reprint requests to: Daizo Hamada, Department of Developmental Infectious Diseases, Research Institute and Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594–1011, Japan; e-mail: daizo{at}lab.mch.pref.osaka.jp; fax: +81-(0)725-57-3021.
(RECEIVED May 30, 2002; FINAL REVISION July 15, 2002; ACCEPTED July 17, 2002)
3 Present address: Department of Developmental Infectious Diseases, Research Institute and Osaka Medical Center for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594–1011, Japan. 
4 Present address: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0217702.
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Abstract |
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5.0 M, close to the concentration of urea corresponding to the midpoint of unfolding (5.3 M). This result indicates that efficient fibril formation involves a balance between the requirement of a significant population of unfolded or partially unfolded molecules and the need to avoid conditions that strongly destabilize intermolecular interactions. Keywords: Amyloid fibril; kinetics; ß-lactoglobulin; thioflavin T; electron microscope; MALDI-TOF mass
Abbreviations: NMR, nuclear magnetic resonances • TEM, transmission electron microscopy • ANS, 1-anilino-8-naphthalene sulfonic acid • ß-LG, ß-lactoglobulin • thioT, thioflavin T • MALDI, matrix associate laser disorption • TOF, time of flight • d-1, day -1
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Introduction |
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Although there are differences in detail between the various morphologies observed for specific amyloid fibrils, analysis of their structures using a range of techniques including X-ray fiber diffraction and electron microscopy reveals common properties of the underlying structure. In particular, the fibrils usually have a diameter ranging from 5 to 20 nm (Vallat et al. 1979; Winer et al. 1979; Merz et al. 1983; Prusiner et al. 1983; Connors et al. 1985), a cross-ß structure consisting of a core of ß-strands (Kirshchner et al. 1987), along with an affinity to bind a range of dyes such as thioflavin T (thioT) (Naiki et al. 1989) and Congo red (Klunk et al. 1999; Westermark et al. 1999). Experimental data also indicate that the formation of amyloid fibrils is a nucleation-dependent process in which initial species produced by the association of specific regions of denatured proteins plays an important role in initiating the process (Jarrett and Lansbury 1993; Lomakin et al. 1996, 1997; Naiki and Nakakuki 1996; Perutz and Windle 2001). The experimental studies suggest that the ability to form amyloid fibrils is a common property of polypeptide chains, although the intrinsic propensities of sequences of amino acids to do so under given conditions will vary widely (Chiti et al. 2002). This observation is potentially of great importance to understand the evolved properties of native proteins, including their folding behavior, and the mechanism in which misfolding and aggregation are normally avoided in correctly functioning living systems (Dobson 2001; Bucciantini et al. 2002).
Bovine ß-lactoglobulin (ß-LG) is one of the major components of the whey of cow's milk. The protein assumes a dimeric structure at neutral pH, but dissociates into monomers at an acidic pH. The conformation of the protein at both neutral and acidic pH has been determined by X-ray crystallography (Brownlow et al. 1997; Qin et al. 1998, 1999) and NMR spectroscopy (Kuwata et al. 1999; Uhrinova et al. 2000). The structures at the different pH values possess the same basic topology, having nine antiparallel ß-strands and one short and one long 
-helix at the carboxyl terminus (Fig. 1
). Although the biological function of ß-LG is still unknown, the protein has the ability to bind to extended hydrophobic compounds such as retinol (Dodin et al. 1990; Dufour et al. 1991; Hambling et al. 1992). Interestingly, the amino acid sequence suggests a significant propensity for -helical structure despite the highly ß-rich fold (Nishikawa and Noguchi 1991). In accord with this observation, an early intermediate state of the protein during refolding has been found to form non-native -helical structures (Hamada et al. 1996; Kuwajima et al. 1996; Hamada and Goto 1997) in the vicinity of the amino-terminal region of the sequence, a region corresponding to the A-strand in the native structure (Kuwata et al. 2001).
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In the present work, we show that fibrillar structures can be readily formed by incubating bovine ß-LG in the presence of urea at pH 7.0 and 37°C for 10–30 days. Transmission electron microscopy (TEM) of the solutions reveals the existence of well-defined fibrils with diameters of 8–10 nm. The solutions enhance the fluorescence of thioT, indicative of the presence of amyloid structures. Furthermore, the addition of aliquots of solutions containing preformed fibrils (i.e., seeding) dramatically reduces the lag time for the initiation of fibril formation and promotes fibril growth. The results indicate that bovine ß-LG is converted into amyloid structures by incubating the protein under conditions where the protein is substantially unfolded but the formation of noncovalent interactions within the ensemble of denatured protein molecules is permitted.
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Results |
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For ß-LG at a concentration of 0.05 mg/mL, a cooperative transition with a midpoint at a urea concentration of 3.1 ± 0.1 M was obtained by monitoring the fluorescence intensity at 343 nm (Fig. 2A, circles). This value is significantly lower than the midpoint of the transition (5.3 ± 0.1 M) obtained from the plot of the peak position of these spectra as a function of urea concentration (Fig. 2B, triangles). The difference in the transition midpoints obtained by fluorescence intensity and peak position suggests that at least one conformational species, other than the native dimer or the fully unfolded state, is present under at least some denaturing conditions.
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).
The hydrophobic dye ANS shows an intense fluorescence signal at 480 nm when it binds to exposed hydrophobic clusters on the surface of partially folded intermediate states of proteins (Semisotnov et al. 1987). In contrast, significant fluorescent changes are rarely observed for ANS molecules in the presence of native or fully unfolded proteins. Exceptionally, native ß-LG stimulates an increase in ANS fluorescence due to its intrinsic property to bind to hydrophobic molecules (Collini et al. 2000). Nevertheless, ANS binding can detect the presence of a partially folded intermediate with non-native -helical structure during the folding and unfolding of this protein at pH 2.0 (D. Hamada and Y. Goto, unpubl.).
In the present work, it has been found that the fluorescence intensity of ANS decreases with increasing concentration of urea (Fig. 2B). The transition midpoint obtained by monitoring this fluorescence change is highly consistent with the unfolding curve obtained from the shift of the intrinsic tryptophan fluorescence peak. This observation suggests that the dissociation of ANS from ß-LG occurs when the protein transforms into its fully unfolded state at high urea concentrations. Although ANS may affect the equilibrium among conformational states, it is rather likely that ANS stabilizes the partially folded states, if it exists, relative to the native state. It is, therefore, highly unlikely that a significant amount of a partially folded species is populated during the urea unfolding of ß-LG at pH 7.0 and 37°C. The same conclusion has been drawn from the analysis of far- and near-UV circular dichroism spectra during unfolding of ß-LG by urea at pH 7.0 and 25°C (M. Yagi, K. Sakurai, and Y. Goto, pers. comm.).
According to these observations, the unfolding transition of ß-LG by urea are considered in the following scheme:
 | (Scheme 1.) |
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GDM,0 = 50.3 ± 0.3 (kJ/mol); mDM = 4.9 ± 0.1 (kJ/mol/M); GMU,0 = 20.1 ± 2.4 (kJ/mol); mMU = 3.8 ± 0.4 (kJ/mol/M). Importantly, the mMU value obtained here at neutral pH was relatively consistent with the value (5.0 ± 0.1 kJ/mol/M; Apenten 1998) obtained at pH 3.0, where the protein predominantly stabilizes the native monomer. This further supports the idea that the conformational state that accumulates during the transition of ß-LG induced by urea is likely to be the native monomer.
Spontaneous fibril formation in urea solutions
The ß-LG solutions containing a variety of concentrations of urea were incubated at 37°C and the existence of amyloid fibrils was probed by TEM and thioT fluorescence.
When solutions of the protein containing 3–5 M urea were incubated for periods of 10–30 days, opalescent precipitates became visible, and the presence of fibrillar aggregates was shown by TEM (Fig. 3). The electron micrographs reveal that these aggregates are unbranched, twisted fibrils with diameters of 8–10 nm (Table 1), comparable to the diameters of typical amyloid fibrils (4–20 nm) (Vallat et al. 1979; Winer et al. 1979; Merz et al. 1983; Prusiner et al. 1983; Connors et al. 1985; Chamberlain et al. 2000). Interestingly, micrographs of the fibrils formed in 3 M urea indicate the presence of some thicker fibrils, with diameters of 15 nm, relative to those formed at the higher urea concentrations.
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). No significant increase in thioT fluorescence was, however, observed for solutions containing 0–2 M urea even after 70 days of incubation. At the protein concentrations used in these experiments (1 mg/mL), ß-LG assumes the native dimer structure at 0–2 M urea, whereas it dissociates into the native monomers at 2–4 M urea before the formation of fibrils (see Fig. 2C). Therefore, it appears that the presence of the native dimer can efficiently prevent the formation and extension of fibril nuclei. This result is consistent with the observation that the stabilization of the native tetramer by substrate binding inhibits the formation of transthyretin fibrils (Miroy et al. 1996).
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). Similar kinetic behavior has been found to occur for many amyloidogenic proteins (Jarrett and Lansbury 1993; Lomakin et al. 1996; Naiki and Nakakuki 1996; Perutz and Windle 2001). Importantly, the urea concentrations showing the largest increase of thioT fluorescence (Fig. 4A) are low (3–5 M) in comparison with the transition region for urea unfolding monitored by intrinsic tryptophan fluorescence (3–7 M) (see Fig. 2). Below the transition region (<3 M urea), the formation of aggregates is efficiently suppressed. Above the transition region where the protein begins to unfold (>3 M urea), the formation of fibrils is clearly observed. As the urea concentration increases, the rate and yield of fibril formation initially increase, reflecting the greater population of unfolded molecules able to aggregate. Above 5 M urea, however, both the rate and yield of fibril formation decrease substantially, a result that can be explained by the increased ability of urea to solvate the unfolded state. This result supports the idea that the fibrillogenesis of proteins is inhibited when noncovalent interactions are destabilized within the ensemble of unfolded structures (Chiti et al. 1999, 2000).
The sigmoidal curves such as these shown in Figure 4B were analyzed by nonlinear least squares curve-fitting to a stretched exponential function: 
(Alvarez et al. 1991; Morozova-Roche et al. 1999; Jund et al. 2000). In this equation, F, F
, and F are the observed fluorescence intensity at time t, the final fluorescence intensity, and the fluorescence amplitude, respectively. ksp is the rate of spontaneous fibril formation. Although the interpretation of the parameters involved in this equation is not straightforward, these parameters are useful in empiric descriptions of complex reactions whose kinetics are not easily modeled (Morozova-Roche et al. 1999; Jund et al. 2000). For example, when the n value is close to 1, the curve can be expressed by a single exponential function with a rate constant of ksp. For 0 < n < 1, the kinetics can be approximated to multiple exponential functions indicative of multiple events. On the other hand, for n > 1, the curves represent a sigmoidal transition with an initial lag phase, suggestive of the involvement of intermediate species. The inverse of the rate constant ksp gives the relaxation time, that is, the time when the 1/e (36.8%) of the reaction is completed (where e is Euler's constant). For n > 1, a reaction becomes more cooperative as the value of n increases.
The F, ksp, and n values determined in this way for the different urea concentrations are shown in Figure 5. Values of n more than 5 were obtained for all the samples showing a significant increase of thioT fluorescence; this result indicates the presence of a lag phase and the relatively high cooperativity of the growth phase that follows. The maximum ksp value (0.058 d-1) was found for the fibril growth in 5.0 M urea. This concentration of urea is close to the midpoint (5.3 M) of the unfolding transition of the monomeric species shown in Figure 2. Thus, at this concentration of urea, there is a substantial population of unfolded protein molecules in which the polypeptide chain is exposed so as to permit efficient aggregation. At higher urea concentrations, the urea molecules can solvate the denatured state and destabilize aggregated species. This effect is likely to reduce significantly the propensity for fibril formation.
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F) of the fluorescence can indicate the approximate quantity of fibrils formed in each solution, because fluorescence intensity is associated with fibril formation and is unlikely to vary substantially with urea concentration under these conditions. As in the case for the ksp value, the absolute value of F is maximum in solutions containing 5.0 M urea. The differences in F for the solutions containing varying urea concentrations suggests that the system is fairly closely balanced between polymerization and depolymerization. Interestingly, the plot of ksp versus F is approximately linear, with a correlation coefficient of 0.94 (Fig. 6). Although a range of factors (e.g., the rates of fibril nucleation, extension, and depolymerization) will affect ksp, it appears to be primarily influenced by the length of the lag phase. As the latter reflects the rate of nucleation in the nucleation-dependent conversion process, the correlation found in Figure 6 implies that the total amount of protein molecules converted into the fibrils increases when the rate of nucleation becomes more rapid. Such a result may reflect the increased probability of the formation of organized fibrils rather than amorphous aggregates.
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In agreement with the nucleation model, the initial lag time for ß-LG fibrillogenesis was found to be significantly diminished when seeding was carried out with aliquots of the solutions incubated at the various concentrations of urea (Fig. 7A). In contrast to the results for spontaneous fibrillogenesis, small but distinctive increases in thioT fluorescence could be monitored even for the solutions containing low concentrations of urea (0–2 M); this finding suggests that spontaneous formation of fibrils can occur under these conditions, but at a very slow rate. The experimental data, therefore, suggest that the conversion of soluble ß-LG into amyloid fibrils in urea proceeds, as in other cases, according to a nucleation-dependent manner. Under ideal conditions, the rate of fibril extension can be obtained by fitting the data to a single exponential function (Naiki and Gejyo 2000). However, the kinetics shown here, even after seeding, are relatively slow and there could be a contribution to the extension kinetics from spontaneously formed nuclei, particularly at the later stage of fibrillogenesis. Therefore, to provide a more accurate description of the rate of intrinsic fibril extension, the initial slope for the reaction (dF/dt)t = 0 was considered in our analysis.
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shows a plot of (dF/dt)t = 0 determined from the rates of fibril formation measured by thioT fluorescence under the seeding conditions as a function of the urea concentration. In this situation, the value of (dF/dt)t = 0 correlates well with the rate of fibril extension. The intrinsic extension rate can be seen to become faster with increasing urea concentrations up to 5 M, but then to decrease as the urea concentration is increased. Interestingly, logarithms of (dF/dt)t = 0 in both the acceleratory and deceleratory regions are approximately linearly dependent on the urea concentration. The results of seeding experiments also indicate that the maximum rate of fibril extension is obtained under conditions where the rate of fibril nucleation is optimum (i.e., 5 M urea) (Figs. 4 and 7). In Figure 8, the values of (dF/dt)t = 0 for the seeding experiment are plotted against the ksp values obtained for spontaneous fibrillogenesis. The plot shows a high correlation between these parameters (the correlation coefficient is 0.96), indicating that the two processes are closely related.
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) (Brownlow et al. 1997). To examine whether such dimeric species are involved in the fibril formation by ß-LG in the presence of urea, and more generally to probe for any degradation of the protein molecules during the time taken for fibril formation to occur, we used matrix associate laser disorption-time of flight (MALDI-TOF) mass spectrometry to characterize ß-LG after its extraction from the fibrillar structures. To find conditions where ß-LG incorporated into the fibrils formed in 5 M urea could be solubilized, we tested a range of denaturants including 8 M urea, 8 M guanidium hydrochloride, and 95% (v/v) acetonitrile/H2O containing 0.05% trifluoroacetic acid; only the acetonitrile/H2O mixture was found to dissolve the fibrils and then only to a limited extent. The mass spectrum obtained for the protein solubilized using this procedure shows the presence of a single species with a molecular mass of 18,432.3 ± 9.2 daltons. This value is close to the molecular mass of monomeric ß-LG (18,463.2 ± 9.2 daltons) determined from a reference spectrum of native ß-LG in water. Thus, it is unlikely that any misfolded dimeric species play a significant role in fibril formation by ß-LG in the presence of urea, or that any significant degradation of the protein takes place during this process.
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Discussion |
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Fibril formation was found to be particularly rapid in the transition region for the unfolding of ß-LG by urea, probably due to the increased population of unfolded protein molecules. However, the rate of formation decreased more than 5 M urea, although the population of unfolded protein molecules increased (Fig. 2C). This observation can be attributed to the need for a high population of unfolded proteins for aggregation to occur and for these unfolded molecules to interact strongly with each other. The former property increases with the concentration of a denaturant such as urea, whereas the latter decreases (Chiti et al. 1999; Bellotti et al. 2000). It is, however, still unclear which conformational species is a direct precursor for fibril formation. There is a possibility for the involvement of partially folded species that accumulates only to a small amount. Such conformational species is more likely to be stabilized under the conditions where the unfolded state is populated and still promote the protein aggregation. As with other amyloid fibrils, the rate of formation was found to be promoted by the addition of preformed fibrils, a finding indicative of a nucleation-dependent process. The present results indicate further that the rate of nucleation correlates well with the rate of fibril extension, a finding suggesting that the interactions required for the nucleation event of fibrillogenesis are similar to those required for fibril extension.
Polymorphism of the structural morphology of amyloid fibrils has been demonstrated for several proteins (Ionescu-Zanetti et al. 1999, Chamberlain et al. 2000; Zurdo et al. 2001). Consistent with these studies, different morphologies were found for fibrils formed at the various urea solutions. In addition, the existence of a variety of network structures composed of filaments with diameters ranging from 4 to 10 nm has been reported for heat-induced ß-LG gels (Hermansson 1986; Langton and Hermansson 1992; Kavanagh et al. 2000). Such polymorphism in the fibril structures formed from a single protein could be caused by differences in the number of protofilaments assembled into the mature fibrils. It could also, however, result from the incorporation in different regions of the sequence of the polypeptide chain with various types of fibrils. Although this latter possibility cannot be eliminated at this stage, the fact that the addition of fibrils formed at 5.0 M urea to the protein solutions at other concentrations of urea successfully promoted fibril extension suggests that the regions of the amino acid sequence that are incorporated into the core region of fibrils are essentially the same in the solutions at different urea concentrations.
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Materials and methods |
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Preparation of seeding solutions
Fibrils for seeding experiments were prepared by centrifuging protein solutions (1 mg/mL) in 5.0 M urea containing fibrils at 20,000 g for 10 min. The pellets were resuspended in water and centrifuged again, and the process repeated twice. The resulting precipitates were resuspended in distilled water, and used for seeding experiments. The amount of protein incorporated into fibrils was determined by measuring the weight of dried protein in the tube after lyophilizing aliquots of the solutions.
Urea unfolding by tryptophan and ANS fluorescence
All experiments were carried out in 10 mM sodium phosphate buffer at pH 7.0 and 37°C. The fluorescence intensity of either tryptophan or ANS was monitored using a F4500 fluorimeter (Hitachi, Tokyo, Japan). For intrinsic tryptophan fluorescence, protein solutions of 0.05 or 1 mg/mL with varying concentrations of urea were excited at 290 nm and the fluorescence at 300–400 nm was monitored. For ANS-binding analysis, solutions of 5 µM ANS in 0.05 mg/mL ß-LG and various concentrations of urea were excited at 398 nm and the fluorescence intensity at 500–600 nm was monitored. For each experiment, the protein sample was incubated for 5 min at 37°C before taking the measurement.
Analysis of the unfolding transitions
The unfolding transition of ß-LG by urea are considered to follow the scheme 1. Thus, the fractions of Ndimer, Nmonomer, and U (fD, fM, and fU, respectively) can be expressed as:
 | ((1)) |
 | ((2)) |
 | ((3)) |
is the total protein concentration (M) in the solution.
Provided that the free energy differences between the dimeric and monomeric native states (GDM) and between the monomeric native and unfolded states (GMU) are dependent on urea concentration, they can be expressed as:
 | ((4)) |
 | ((5)) |
GDM,0 and GMU,0 are GDM and GMU in the absence of urea, mDM and mMU are the measures of cooperativity of transitions between the dimeric and monomeric native states and between the monomeric native and unfolded states, respectively. GDM,0, GMU,0, mDM, and mMU were calculated by full deconvolution of all the tryptophan fluorescence spectra obtained at different urea concentrations according to the functions shown in eqs. 1–5.
Thioflavin T assay
Solutions (1.2 mL) of 1 mg/mL protein and various concentrations of urea were prepared in 1.5-mL plastic tubes with lids, and the tubes were then tightly sealed to prevent evaporation. The samples were placed in an air incubator at 37°C. For each incubation time, 40-µL aliquots of the solutions were taken and mixed with 360 µL of 5 µM thioT solution in 10 mM sodium phosphate buffer at pH 7.0. The fluorescence at 465–665 nm was monitored using 5-mm cuvettes. The excitation wavelength was 450 nm. A stretched exponential function, expressed as 
, was used in the curve-fitting analysis of the kinetics of spontaneous fibril formation determined from thioT fluorescence. The ksp and n values are the rate constant (day-1) and heterogeneity parameter, respectively (Morozova-Roche et al. 1999; Jund et al. 2000). The F and F values are the fluorescence intensity at the end of reaction and the amplitude change during the reaction. For the seeding experiments, 10 µg of aliquots of preformed fibrils were added to the protein solutions (1 mg/mL) containing various amounts of urea. The initial rates of fibril extension were determined to eliminate possible errors due to spontaneous nucleation.
TEM imaging
TEM images of samples showing a typical fluorescence increase by thioT were acquired with a JEM 1010 or JEM-1200EX II transmission electron microscope (JEOL, Tokyo, Japan). The acceleration voltage was 80 or 85 kV. The samples were negatively stained by either uranium acetate or phosphotungstate.
Mass spectroscopy
Mass spectra of resolubilized fibrils formed by ß-LG were obtained using a Voyager DE MALDI-TOF mass spectrometer from PerSeptive Biosystems (Framingham, Massachusetts, USA). Sinapinic acid was used to form the matrix complex. Fibrils prepared as described above were resolubilized using 95% acetonitrile/5% H2O, containing 0.05% trifluoroacetic acid.
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Acknowledgments |
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) or 1.0 (
) mg/mL of ß-LG. The ordinate is the relative value to the fluorescence of unfolded protein in 8.0 M urea. (B) Data obtained by monitoring the frequency shift of the fluorescence maximum in the presence of 0.05 (
) and 1.0 () mg/mL of ß-LG, and the fluorescence intensity of ANS (
) in the presence of 0.05 mg/mL of ß-LG. (C) Fractions of native dimeric, native monomeric, and unfolded states as a function of urea concentration in the presence of 1 mg/mL of ß-LG. The curves were drawn using the parameters shown in the text.
), 1 (
), 2 (
), 3 (