The Structure of Denatured alpha -Lactalbumin Elucidated by the Technique of Disulfide Scrambling

FRACTIONATION OF CONFORMATIONAL ISOMERS OF alpha -LACTALBUMIN*

Jui-Yoa ChangDagger and Li Li

From the Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, Houston, Texas 77030

Received for publication, November 27, 2000, and in revised form, December 14, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The structure of denatured alpha -lactalbumin (alpha -LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha -LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha -LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha -LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha -LA. Most importantly, unique isomers of partially unfolded alpha -LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha -helical domain, generating a predominant isomer containing two native disulfide bonds at the beta -sheet domain and two scrambled disulfide bonds at the alpha -helical region. Thermal denaturation selectively unfolds the beta -sheet domain of alpha -LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha -LA.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -LA1 represents one of the most extensively investigated models for understanding the mechanism of protein stability, folding, and unfolding. Under a variety of conditions, alpha -LA adopts a partially structured conformation termed as "molten globule" (1-9), which has been observed along both the unfolding (1, 2, 5, 10-12) and folding pathways (13-18) of alpha -LA and is considered one of the best characterized folding/unfolding intermediates of globular proteins. At low pH (1, 19, 20), elevated temperature (19, 21), or mild concentration of denaturant (e.g. GdmCl; Refs. 22, 23), the native alpha -LA unfolds to the state of a molten globule. During the refolding, the fully denatured alpha -LA undergoes a rapid collapse to the state of molten globule (13, 14, 18) as intermediate, which is followed by consolidation and search of side chain interactions to attain the native protein (14). The structure of alpha -LA molten globule is characterized by a high degree of native-like secondary structure and a fluctuated tertiary fold (1-4, 19). It is stabilized mainly by the core hydrophobic amino acids within the alpha -helical domain (24-28) and may form even in the absence of its four disulfide bonds (15). The structure of the molten globule of alpha -LA is also highly heterogeneous (3, 27, 29). Like most denatured or partially denatured state of proteins (30, 31), it comprises a large number of conformational isomers. So far, characterization of the structure of denatured alpha -LA and the molten globule state of alpha -LA have been achieved by measuring the average property of these collective isomers using a wide range of spectroscopic and physiochemical methods, including circular dichroism (1, 2, 19, 32), fluorescence (12, 32, 33), NMR (5, 14, 15, 20, 29, 34), disulfide replacement (35-37), limited proteolysis (38, 39), light scattering (40, 41), and calorimetric techniques (42). Further understanding of the denatured state of alpha -LA and the molten globule state of alpha -LA will require fractionation of diverse populations of conformational isomers that constitute the denatured alpha -LA.

In this report, the structure of denatured alpha -LA has been analyzed by the technique of disulfide scrambling (43), which permits fractionation and quantitative analysis of the denatured isomers. In the presence of denaturant and thiol initiator, the native alpha -LA denatures by shuffling its native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers that are trapped by non-native disulfide bonds. alpha -LA contains four disulfide bonds and may adopt 104 possible scrambled isomers. The technique of disulfide scrambling (43) presents numerous advantages for characterizing the structure of denatured alpha -LA. 1) Scrambled isomers of alpha -LA are not interconvertible in the absence of thiol catalyst or acidic pH. Because of their stability and diverse physicochemical properties, scrambled isomers of alpha -LA can be separated and purified by liquid chromatography and structurally characterized (43-46). This permits a more detailed description of the structure of denatured alpha -LA that is not attainable with conventional methods. 2) Scrambled isomers of alpha -LA contain different sizes of disulfide loops and adopt varied degree of unfolding. Compositional analysis of scrambled isomers allows evaluation of the extent of unfolding of the denatured alpha -LA (43, 44, 46). 3) The process of denaturation of alpha -LA can be monitored in a time-course manner by acid trapping of unfolding intermediates. This permits identification and isolation of structurally defined intermediates present along the unfolding pathway of alpha -LA. Specifically, it will allow us to examine the structure of scrambled isomers that constitute the molten globule state of alpha -LA.

Our specific aims are as follows: 1) to analyze the structure and heterogeneity of denatured alpha -LA, 2) to elucidate the unfolding pathway of alpha -LA denatured under increasing concentrations of selected denaturants, and 3) to characterize the molecular structure of the molten globule state of alpha -LA.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Calcium-depleted bovine alpha -LA (L-6010) was used throughout this study and was obtained from Sigma. The protein was further purified by HPLC and was shown to be more than 97% pure. GdmCl, GdmSCN, urea, acetonitrile, and 2-mercaptoethanol were also purchased from Sigma with a grade purity of greater than 99%.

Denaturation of the Native alpha -LA-- The native alpha -LA (0.5 mg/ml) was dissolved in Tris-HCl buffer (0.1 M), pH 8.4, containing 2-mercaptoethanol (0.02-0.25 mM) and selected concentrations of denaturants (urea, GdmCl, GdmSCN, or acetonitrile). Denaturation was typically performed at 23 °C for 24-48 h. In the case of thermal denaturation, the sample (in the presence of 0.1 mM 2-mercaptoethanol) was subjected to elevated temperature (45-65 °C) for a time period of up to 60 min. To monitor the kinetics and intermediates of unfolding, aliquots of the sample were removed in a time-course manner, quenched with an equal volume of 4% aqueous trifluoroacetic acid, and analyzed by reversed-phase HPLC. The denatured sample was subsequently acidified with 4% trifluoroacetic acid and stored at -20 °C.

Criteria for the State of Equilibrium and the End Point of Denaturation-- The end point of denaturation, under a given denaturing condition, implies that conversion of the native species to the scrambled isomers has reached a state of equilibrium. This state of equilibrium also includes those among scrambled isomers. The major criteria to ensure that denaturation has reached equilibrium is time-course kinetics analysis, which indicates that conversion of the native alpha -LA to the scrambled species has reached a plateau, and the ratio of scrambled to the native species remains constant during prolonged sample incubation. Another method to verify the state of equilibrium of denaturation is to renature (refold) the extensively unfolded alpha -LA under the same conditions that denature the native alpha -LA, for instance, by allowing the sample denatured with 8 M GdmCl to refold after dilution of the denaturant. Both renaturation and denaturation are expected to reach the same state of equilibrium and generate end products that consist of the same ratio of scrambled to native species (49). A systematic study has shown that denaturation of alpha -LA by denaturants (in the presence of 0.1-0.25 mM 2-mercaptoethanol) completed generally within 2 h (see Fig. 2). Nonetheless, the reaction has been routinely allowed to proceed overnight (24 h). On the other hand, completion of denaturation of alpha -LA by acetonitrile is relative slow and requires about 15 h. In the case of thermal denaturation, there are inherent risks of prolonged sample incubation because of the heat-induced decomposition of disulfide bonds. Experiments of thermal denaturation of alpha -LA were therefore performed only in a time-course manner within 60 min.

Denaturation Is Distinguished from Unfolding-- The denatured alpha -LA may adopt varied degrees of unfolding. Denaturation and unfolding are therefore two distinct terms. Using the method of disulfide scrambling (43), it is possible to observe and follow simultaneously the process of denaturation and unfolding of alpha -LA. The extent of denaturation of alpha -LA is defined by the simple conversion of the native structure to non-native structures (scrambled isomers). There are 104 scrambled isomers of denatured alpha -LA versus one isomer of the native alpha -LA. Unfolding of alpha -LA is defined by the state of denatured alpha -LA and is structurally characterized by the composition (relative concentration) among the 104 scrambled isomers.

Plotting of the Kinetic and Thermodynamic Denaturation Curves of alpha -LA-- The denaturation curve of alpha -LA was determined by the fraction (%) of the native alpha -LA converted to the scrambled isomers. Quantitative analysis of the relative yield of scrambled and the native isomer was based on the integration of HPLC peak areas. There are two types of denaturation curves: 1) kinetic denaturation curve was calculated and derived from samples denatured in a time-course manner under fixed denaturing conditions, and 2) thermodynamic denaturation curve was derived from those that have attained the end point (equilibrium) of denaturation under increasing concentrations of a selected denaturant.

Plotting of the Kinetic and Thermodynamic Unfolding Curves of alpha -LA-- The unfolding curve of alpha -LA was determined by the ebb and flow of different scrambled isomers as fractions of the total denatured (scrambled) protein along the pathway of unfolding. Similar to the denaturation curve, the unfolding curve can be established as follows: 1) from samples denatured in a time-course manner by a defined denaturing condition (kinetic unfolding curve) or 2) from samples that have attained the end point of denaturation under increasing concentrations of a selected denaturant (thermodynamic unfolding curve). Calculation of the yield of scrambled isomers was based on the peak area integration. Because of the complexity of minor isomers, the data have a S.D. of ± 5%.

Structural Analysis of Scrambled Isomers of alpha -LA-- Fractions of scrambled alpha -LA (~10 µg) were isolated and treated with 1 µg of thermolysin in 30 µl of N-ethylmorpholine/acetate buffer (50 mM), pH 6.4. Digestion was carried out at 37 °C for 16 h. Peptides were then isolated by HPLC and analyzed by amino acid sequencing and mass spectrometry to identify the disulfide-containing peptides.

Amino Acid Sequencing and Mass Spectrometry-- Amino acid sequence of disulfide-containing peptides were analyzed by automatic Edman degradation using a PerkinElmer Procise sequencer (Model 494) equipped with an online PTH-derivative amino acid analyzer. The molecular mass of peptides was determined by MALDI-TOF mass spectrometry (Perkin-Elmer Voyager-DE STR).

Nomenclature of Scrambled Isomers of alpha -LA-- Scrambled species of alpha -LA are designated by the following formula: X-alpha -LA-(species assigned on HPLC), where X stands for scrambled. For instance, X-alpha -LA-a represents species "a" of scrambled alpha -LA.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Disulfide Structures of the Predominant Isomers of Denatured alpha -LA-- Denatured alpha -LA was shown to consist of at least 45 fractions of fully oxidized scrambled isomers. Their relative concentrations vary substantially under different denaturing conditions (see the following sections). Among them, six major fractions of scrambled alpha -LA were isolated and structurally characterized. They are X-alpha -LA-a, X-alpha -LA-b, X-alpha -LA-c, X-alpha -LA-d, X-alpha -LA-e, X-alpha -LA-h. After digestion with thermolysin, peptides were isolated by HPLC and were characterized by Edman sequencing and MALDI mass spectrometry to identify the peptides that contain disulfides (data available upon request). The disulfide structures are given in Fig. 1. X-alpha -LA-a and X-alpha -LA-d contain, among the 104 possible isomers, the smallest sizes of disulfide loops and represent the most extensively unfolded structures of alpha -LA. These two isomers become well populated under strong denaturing conditions. Specifically, X-alpha -LA-a adopts the beads-form structure with disulfides formed by four consecutive pairs of neighboring cysteines. X-alpha -LA-b and X-alpha -LA-c are partially unfolded isomers, with X-alpha -LA-b possessing two native disulfide bonds within the beta -sheet region and X-alpha -LA-c retaining two intact native disulfide bonds at the alpha -helical domain.


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Fig. 1.   The structures of native and denatured alpha -LA. Top, three-dimensional structure of the native alpha -LA and schematic presentation of four denatured scrambled isomers (a, b, c, and d). The dark gray and light gray indicate the sections of alpha -helical and beta -sheet structures, respectively. Bottom, disulfide structures of the native alpha -LA and six scrambled isomers.

Structures of alpha -LA Denatured by Urea, GdmCl, and GdmSCN-- Time-dependent kinetics denaturation of the native alpha -LA was first analyzed using 6 M urea and 4 M GdmCl in the presence of varying concentrations of thiol initiator (0.02-0.25 mM 2-mercaptoethanol). These experiments were designed to establish conditions that would permit denaturation of alpha -LA to reach a state of equilibrium. The results are shown in Fig. 2. Both urea and GdmCl denature the native alpha -LA at similar rates (Fig. 2, right column). In the presence of 0.1-0.25 mM 2-mercaptoethanol, denaturation of alpha -LA is completed within 2 h. The structure of alpha -LA denatured by 6 M urea comprises 4 major scrambled isomers (Fig. 2, left column).


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Fig. 2.   Kinetic denaturation of alpha -LA by urea (6 M) and GdmCl (4 M). Denaturation was carried out at 23 °C in Tris-HCl buffer, pH 8.4 containing varying concentrations of 2-mercaptoethanol (0.02-0.25 mM). Intermediates of denaturation were trapped by acidification (4% trifluoroacetic acid) and analyzed by HPLC, using the conditions described in the legend to Fig. 3. Left, chromatograms of time course-trapped intermediates denatured by 6 M urea in the presence of 0.1 mM 2-mercaptoethanol. Right, effect of the concentration of 2-mercaptoethanol (inset) on the kinetic denaturation curves of alpha -LA. Fractions denatured indicate the fraction (%) of native alpha -LA converted to scrambled alpha -LA.

Thermodynamic denaturation of alpha -LA was subsequently performed in the presence of 2-mercaptoethanol (0.1 mM) and increasing concentrations of urea, GdmCl and GdmSCN. Chromatograms of the denatured alpha -LA are shown in Fig. 3. At mild concentrations of GdmCl (1.75 M) and GdmSCN (0.75 M), denatured alpha -LA was shown to consist of at least 45 fractions of scrambled isomers. Under strong denaturing conditions, the denatured alpha -LA comprises three predominant isomers (X-alpha -LA-a, X-alpha -LA-b, and X-alpha -LA-d). The thermodynamic denaturation curves (Fig. 4) reveal that GdmSCN is about 2- and 7-fold more potent than GdmCl and urea, respectively. In the presence of CaCl2 (5 mM), the potency of GdmCl and urea to denature the native alpha -LA was diminished by 2-fold. The thermodynamic unfolding curves (Fig. 5) further display the structures of denatured alpha -LA evolved under increasing concentrations of selected denaturants. Characteristically, the progressive unfolding of denatured alpha -LA is accompanied by an increasing yield of X-alpha -LA-a. For instance, the recovery of X-alpha -LA-a as a fraction of the total scrambled alpha -LA increases from 5 to 27% as the concentration of GdmSCN rises from 0.2 to 4 M (Fig. 5). Similar phenomenon was observed with the urea and GdmCl denaturation of alpha -LA.


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Fig. 3.   Thermodynamic denaturation of alpha -LA by different concentrations of urea, GdmCl, and GdmSCN. Denaturation was carried out at 23 °C for 24 h in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol (0.1 mM). Denatured samples were acidified with 4% trifluoroacetic acid and analyzed by reversed-phase HPLC using the following conditions. Solvent A for HPLC was water containing 0.1% trifluoroacetic acid. Solvent B was acetonitrile/water (9:1 v/v) containing 0.086% trifluoroacetic acid. The gradient was 22-37% B in 15 min and 37-56% B from 15 to 45 min. The flow rate was 0.5 ml/min. Column was Zorbax 300SB C18 for peptides and proteins, 4.6 mm × 5 µm. Column temperature was 23 °C. The concentration of denaturant and predominant isomers (a-h) of denatured alpha -LA are marked.


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Fig. 4.   Thermodynamic denaturation curves of alpha -LA. These curves were derived from data presented in Fig. 3. In the case of GdmCl and urea denaturation, experiments were also performed in the presence of CaCl2 (5 mM). Fractions denatured indicates the fraction (%) of native alpha -LA converted to scrambled alpha -LA.


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Fig. 5.   Thermodynamic unfolding curves of alpha -LA. These curves were derived from data presented in Fig. 3. The recoveries of seven major scrambled isomers were used to construct these curves.

Structures of alpha -LA Denatured by Organic Solvent-- Kinetic denaturation of alpha -LA by organic solvent was first carried out in Tris-HCl buffer (0.1 M), pH 8.4, containing 40% acetonitrile. The experiments were performed both in the absence and presence of CaCl2 (5 mM, Fig. 6). The results show that the rate of disulfide shuffling induced by acetonitrile is about 10-fold slower than those promoted by urea and GdmCl (Fig. 2). The reaction reaches equilibrium after about 15 h of incubation. In the absence of CaCl2 (Fig. 6, left column), unfolding of denatured alpha -LA proceeds through X-alpha -LA-c as a predominant intermediate, and the denatured sample consists of two major scrambled isomers, X-alpha -LA-a and X-alpha -LA-b. In the presence of CaCl2 (Fig. 6, right column), the yield of X-alpha -LA-c as unfolding intermediate diminishes drastically, and the denatured alpha -LA comprises one prevalent isomer, X-alpha -LA-b.


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Fig. 6.   Kinetic denaturation of alpha -LA by acetonitrile. Denaturation was carried out at 23 °C in Tris-HCl buffer, pH 8.4 containing acetonitrile (7.28 M or 40% by volume) and of 2-mercaptoethanol (0.1 mM). Intermediates of denaturation were trapped by an equal volume of 4% trifluoroacetic acid and analyzed by HPLC using the conditions described in the legend to Fig. 3. Left, denaturation carried out in the absence of CaCl2. Right, denaturation performed in the presence of CaCl2 (5 mM).

Thermodynamic denaturation of the native alpha -LA was then performed with increasing concentrations of acetonitrile (10-50%, by volume). All reactions were allowed for 48 h to ensure the state of equilibrium. The experiments were also conducted in the absence and presence of CaCl2, and the results are presented in Fig. 7. Abrupt denaturation of alpha -LA occurs at between 20 and 30% (1.82-3.64 M) acetonitrile. The thermodynamic unfolding curves demonstrate that in the absence of CaCl2, unfolding of denatured alpha -LA is characterized by an increasing yield of both X-alpha -LA-a and X-alpha -LA-b. (Fig. 7A). In the presence of CaCl2, the structure of denatured alpha -LA is dominated by the isomer X-alpha -LA-b (Fig. 7B).


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Fig. 7.   Thermodynamic denaturation of alpha -LA by acetonitrile. Denaturation was carried out at 23 °C in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol (0.1 mM) and different concentrations of acetonitrile (10-50% by volume, equivalent to 1.82-9.10 M in molar concentration). Denaturation proceeded for 48 h. Denatured samples were acidified with 4% trifluoroacetic acid and analyzed by HPLC. Left, chromatograms obtained from denaturation carried out in the absence of CaCl2. The corresponding thermodynamic unfolding curves are given in A. Right, chromatograms obtained from denaturation performed in the presence of CaCl2 (5 mM). The corresponding unfolding curves are given in B.

Thermal Denaturation of alpha -LA-- Thermal denaturation of disulfide-containing proteins can be practically performed only in a kinetic fashion. This is because prolonged incubation at high temperature in alkaline pH may lead to the decomposition of the disulfide bonds of scrambled isomers. The native alpha -LA was therefore denatured at 45-65 °C for up to 60 min. The rate of alpha -LA denaturation increases by 3- and 5-fold as the temperature rises from 45 to 55 °C and 65 °C, respectively (Fig. 8). At 65 °C, the presence of CaCl2 (5 mM) reduces the rate of denaturation by a factor of about 3-fold (Fig. 8).


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Fig. 8.   Kinetic denaturation curves of alpha -LA acquired from the experiments of thermal denaturation. Denaturation was carried out at 45, 55, and 65 °C in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol (0.1 mM). Denaturation at 65 °C was also performed in the presence of CaCl2 (5 mM). Fractions denatured indicate the fraction (%) of native alpha -LA converted to scrambled alpha -LA.

Heat-denatured alpha -LA comprises many of the isomers found within the structures of alpha -LA denatured by GdmCl and GdmSCN (Fig. 9). The most striking feature is the prevalence of X-alpha -LA-c during the early stage of thermal denaturation. Under selected denaturing conditions (e.g. 65 °C, 2 min), the yield of X-alpha -LA-c may account for as much as 40% of the total content of the denatured alpha -LA. This is clearly demonstrated by the kinetic unfolding curves of thermal denaturation of alpha -LA (Fig. 9A). The inclusion of CaCl2, which binds to the beta -sheet domain of alpha -LA, inhibits not only the rate of thermal denaturation (Fig. 8) but also the prevalence of X-alpha -LA-c as the unfolding intermediate (Fig. 9, B and top right HPLC chromatograms).


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Fig. 9.   Kinetic denaturation of alpha -LA by elevated temperature. Denaturation was carried out at 65 °C in Tris-HCl buffer, pH 8.4 containing 2-mercaptoethanol (0.1 mM). The experiments were conducted both in the absence (left chromatograms) and presence (right chromatograms) of CaCl2. Unfolding intermediates were trapped by acidification (4% trifluoroacetic acid) and analyzed by HPLC using the conditions described in the legend of Fig. 3. Predominant isomers of denatured alpha -LA are marked. The corresponding unfolding curves are presented in A and B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Structure of Denatured alpha -LA Is Highly Heterogeneous-- Structural analysis of denatured proteins is inherently difficult. Unlike the native protein, which usually adopts a well defined structure, a denatured protein comprises highly heterogeneous conformational isomers that generally exist in a state of equilibrium (30, 31). It is a daunting task to attempt isolation and analysis of denatured conformational isomers, not only because of the exceedingly large number of isomers that may exist, but also because of their instability and rapid interconversion. Without isolation of conformational isomers, the structural property of a denatured protein has been typically concluded from the measurement of the collective isomers using various spectroscopic techniques (47-51). This has been the case for the analysis of most denatured proteins, including alpha -LA.

The technique of disulfide scrambling permits isomers of a denatured protein to be trapped by non-native disulfide bonds. Such stabilized isomers can be separated by liquid chromatography and structurally characterized (43). For a protein that contains four disulfide bonds, like alpha -LA, there exist 104 possible species of scrambled isomers. Denatured alpha -LA was shown to comprise about 50% of these possible isomers. The heterogeneity is dependent upon the denaturing conditions and is most evident for samples denatured at elevated temperature (Fig. 9) or low concentrations of denaturants (Fig. 3). A similar extent of structural heterogeneity was observed with several three-disulfide models. In the case of hirudin (45), tick anticoagulant peptide (43), and potato carboxypeptidase inhibitor (46), about 50-80% of the possible scrambled isomers were demonstrated to constitute their denatured structures.

Conformational Stability of alpha -LA Is Characterized by Its Denaturation Curves-- Denaturation curves of alpha -LA have been determined here using the technique of disulfide scrambling (Fig. 4). They are comparable with those evaluated by the CD signal of alpha -LA (1). Both techniques reveal that alpha -LA (calcium-depleted) denatures precipitously at 1-2 M GdmCl. The signal for disulfide shuffling is as sensitive as that of fluorescence or CD spectra (49) in measuring the denaturation curves of proteins. This was demonstrated in the case of RNase A (52) and potato carboxypeptidase inhibitor (46).

Aside from alpha -LA, conformational stability of seven different disulfide proteins were similarly investigated using the technique of disulfide scrambling. Their relative stability is presented in Table I. The comparison is defined by the concentration of a denaturant required to achieve 50% of the protein denaturation thermodynamically. Among them, calcium-depleted alpha -LA represents the least stable protein. It is about 2-fold less stable than RNase A, a protein also containing four disulfide bonds and having a size almost identical to that of alpha -LA. However, calcium-bound alpha -LA exhibits stability similar to that of RNase A (Table I). The following can be concluded from evaluation of this data. 1) These results demonstrate and confirm that GdmSCN is typically 2-3-fold more potent than GdmCl, which in turn is an additional 2-3-fold more effective than urea (49). 2) Ranking of protein stability depends upon the nature of denaturant. For example, based on the GdmSCN denaturation curves, IGF-1 is 1.5-fold more stable than tick anticoagulant peptide. This order is reversed when comparison of their stability is based on GdmCl denaturation curves (Table I). 3) Numerous proteins are barely denatured by M urea, a condition that is general considered as being capable to fully denature proteins (31). The most remarkable case is BPTI, which remains practically intact at 8 M urea (53). For hirudin and EGF, less than 15% is denatured under the same condition (45).

                              
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Table I
The concentration of denaturant required to denature 50% of the protein

The Mechanism of Denaturation of alpha -LA Is Illustrated by Its Unfolding Curves-- The unfolding curves of alpha -LA are derived from the structural analysis of denatured alpha -LA, which is characterized by the composition of its scrambled species. The structure of denatured alpha -LA varies, depending on the kinetics of denaturation, on the nature and strength of denaturant. Unfolding curves of alpha -LA thus can be obtained as follows: 1) from structural analysis of samples denatured by a fixed denaturing condition and quenched in a time-course manner (Fig. 9, kinetic unfolding curves) or 2) from samples that have reached equilibrium of denaturation under increasing concentrations of a selected denaturant (thermodynamic unfolding curves, see Figs. 5 and 7). These unfolding curves of alpha -LA, constructed from the relative recoveries of seven major scrambled isomers, display two important features about the mechanism of denaturation of alpha -LA.

First, it demonstrates a general pathway for the unfolding of denatured alpha -LA-a involving progressive expansion and relaxation of the protein conformation toward the shape of a linear structure. Among the 104 possible scrambled isomers of alpha -LA, X-alpha -LA-a (beads-form isomer) and X-alpha -LA-d contain the smallest disulfide loops and presumably represent the most extensively unfolded structures. The yield of X-alpha -LA-a and X-alpha -LA-d are relative to the strength of the denaturing condition. This phenomenon is most evident with samples denatured with urea, GdmCl, GdmSCN, and acetonitrile. It can be measured quantitatively from thermodynamic unfolding curves presented in Figs. 5 and 7A. As the concentration of urea increases from 3 to 8 M, the recovery of X-alpha -LA-a as a fraction of the total denatured protein grows from 9 to 32%. The same tendency was found with samples denatured by GdmCl and GdmSCN. Between 1 and 6 M GdmSCN, the recovery of X-alpha -LA-d rises from 3 to 23%. alpha -LA is not alone in displaying this property. The predominance of beads-form isomer under strong denaturing conditions was similarly observed with the unfolding behaviors of tick anticoagulant peptide (43), insulin-like growth factor (44), hirudin (45), potato carboxypeptidase inhibitor, (46) and bovine pancreatic trypsin inhibitor (53). For example, in the presence of 6 M GdmSCN, more than 63% of the denatured tick anticoagulant peptide and 55% of denatured BPTI were found to be the beads-form isomer. In the case of IGF-1, the content of the beads-form isomer also rises from 5 to 30% as the concentration of GdmSCN increases from 1 to 6 M.

Second, it reveals the presence of a partially structured unfolding intermediate, which exhibits structural characteristics of the molten globule state of alpha -LA. One of the scrambled isomers, X-alpha -LA-c was found to contain two native disulfide bonds (Cys6-Cys120 and Cys28-Cys111) within the alpha -helical domain and two non-native disulfide bonds (Cys61-Cys73 and Cys77-Cys91) within the beta -sheet (calcium-binding) domain (Fig. 1). The disulfide structure of X-alpha -LA-c implies that it is a partially unfolded alpha -LA with a largely intact alpha -helical domain and an unstructured, disordered beta -sheet region. X-alpha -LA-c accumulates only under mild denaturing conditions. At low concentrations of GdmCl (1 M) and GdmSCN (0.2 M, see Fig. 5), X-alpha -LA-c constitutes ~10% of the total denatured alpha -LA. However, prevalence of X-alpha -LA-c as an unfolding intermediate is most obvious with thermal denaturation (Fig. 9) or acetonitrile as denaturant (Fig. 6). In both cases, X-alpha -LA-c may account for more than 40% of the denatured alpha -LA during the early stages of kinetic denaturation.

The disulfide structure of X-alpha -LA-c is consistent with the properties of well characterized molten globule of alpha -LA. Structural elements that stabilize and characterize the structure of molten globule of alpha -LA have been investigated by many different laboratories (15-17, 24, 25, 29, 33, 37). NMR analysis showed that the most persistent structure in the alpha -LA molten globule is localized at the helical domain. Kim and co-workers (29) have demonstrated that the molten globule properties of alpha -LA are mainly confined to one of its two domains. The alpha -helical domain forms a helical structure with a native-like tertiary fold, whereas the beta -sheet domain is essentially disordered (24). These new findings differ somehow from the common definition of molten globule in which a fluctuated tertiary fold is believed to encompass the entire polypeptide chain (1, 2). Our data support the finding that the molten globule of alpha -LA is composed of a structured and an unstructured domain.

Binding of Ca2+ Stabilizes the beta -Sheet Domain of alpha -LA-- Binding of Ca2+ is known to stabilize the conformation of alpha -LA (for review, see Ref. 54). Our data confirm this phenomenon. The GdmCl denaturation curves show that the stability of alpha -LA increases by 2-fold in the presence of 5 mM CaCl2 (Fig. 4). A similar outcome was observed when alpha -LA was denatured by acetonitrile in a thermodynamic fashion (Fig. 7). Binding of Ca2+ to alpha -LA also impedes the initial rate of its thermal denaturation by a factor of 3 (Fig. 8). Surprisingly, the binding of Ca2+ does not decelerate the kinetics of alpha -LA denaturation by acetonitrile (40%) (Fig. 6).

Structural analysis of denatured alpha -LA reveals that binding of Ca2+ protects the two native disulfide bonds (Cys61-Cys77 and Cys73-Cys91) located within the beta -sheet domain of alpha -LA. This is displayed by thermal denaturation of alpha -LA (Fig. 9) in which the binding of Ca2+ inhibits disulfide scrambling of Cys61-Cys77 and Cys73-Cys91 and suppresses the recovery of X-alpha -LA-c. However, this effect is most remarkable in the case of denaturation by acetonitrile. For example, alpha -LA denatured in the presence of 30% acetonitrile and CaCl2 (5 mM) comprises a single predominant scrambled isomers (X-alpha -LA-b) that retains two intact native disulfide bonds of the beta -sheet domain (Fig. 7). These data are consistent with the defined calcium binding site of alpha -LA, which is located at the beta -domain. It is formed by oxygen ligands from carboxylic groups of Asp82, Asp87, and Asp88, and two carbonyl groups of the peptide backbone (residues 79 and 84; Refs. 54, 55).

    FOOTNOTES

* This work was supported by an endowment from the Robert Welch Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Inst. of Molecular Medicine, 2121 W. Holcombe Blvd. Houston, Texas 77030. Tel.: 713-500-2458; Fax: 713-500-2424; E-mail: Rowen.Chang@uth.tmc.edu.

Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M010700200

    ABBREVIATIONS

The abbreviations used are: alpha -LA, alpha -lactalbumin; X-alpha -LA, scrambled alpha -lactalbumin; GdmCl, guanidine hydrochloride; GdmSCN, guanidine thiocyanate; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Kuwajima, K. (1989) Proteins Struct. Funct. Genet. 6, 87-103[Medline] [Order article via Infotrieve]
2. Ptitsyn, O. B. (1995) Adv. Protein Chem. 47, 83-229[Medline] [Order article via Infotrieve]
3. Kuwajima, K. (1996) FASEB J. 10, 102-109[Abstract/Free Full Text]
4. Ptitsyn, O. B. (1995) Trends Biochem. Sci. 20, 376-937[CrossRef][Medline] [Order article via Infotrieve]
5. Barrick, D., and Baldwin, R. L. (1993) Protein Sci. 2, 869-876[Abstract/Free Full Text]
6. Ewbank, J. J., Creighton, T. E., Hayer-Hartl, M. K., and Hartl, F. (1995) Nat. Struct. Biol. 2, 10-11[Medline] [Order article via Infotrieve]
7. Ptitsyn, O. B., and Uversky, V. N. (1994) FEBS Lett. 341, 15-18[CrossRef][Medline] [Order article via Infotrieve]
8. Pande, V. S., and Rokhsar, D. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1490-1494[Abstract/Free Full Text]
9. Pfeil, W. (1998) Proteins 30, 43-48[CrossRef][Medline] [Order article via Infotrieve].
10. Ewbank, J. J., and Creighton, T. E. (1993) Biochemistry 32, 677-693
11. Vanderheeren, G., Hanssens, I., Meijberg, W., and Van Aerschot, A. (1996) Biochemistry 35, 16753-16759[CrossRef][Medline] [Order article via Infotrieve]
12. Gussakovsky, E. E., and Haas, E. (1995) Protein Sci. 4, 2319-2326[Abstract/Free Full Text]
13. Kuwajima, K., Semisotnov, G. V., Finkelstein, A. V., Sugai, S., and Ptitsyn, O. B. (1993) FEBS Lett. 334, 265-268[CrossRef][Medline] [Order article via Infotrieve]
14. Forge, V., Wijesinha, R. T., Balbach, J., Brew, K., Robinson, C. V., Redfield, C., and Dobson, C. M. (1999) J. Mol. Biol. 288, 673-688[CrossRef][Medline] [Order article via Infotrieve]
15. Redfield, C., Schulman, B. A., Milhollen, M. A., Kim, P. S., and Dobson, C. M. (1999) Nat. Struct. Biol. 6, 948-952[CrossRef][Medline] [Order article via Infotrieve]
16. Luo, Y., and Baldwin, R. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11283-11287[Abstract/Free Full Text]
17. Creighton, T. E. (1997) Trends Biochem. Sci. 22, 6-10[CrossRef][Medline] [Order article via Infotrieve]
18. Arai, M., and Kuwajima, K. (1996) Fold Des. 1, 275-287[Medline] [Order article via Infotrieve]
19. Dolgikh, D. A., Gilmanshin, R. I., Brazhnikov, E. V., Bychkova, V. E., Semisotnov, G. V., Venyaminov, S., and Ptitsyn, O. B. (1981) FEBS Lett. 136, 311-315[CrossRef][Medline] [Order article via Infotrieve]
20. Kim, S., and Baum, J. (1998) Protein Sci. 7, 1930-1938[Abstract/Free Full Text]
21. Vanderheeren, G., and Hanssens, I. (1994) J. Biol. Chem. 269, 7090-7094[Abstract/Free Full Text]
22. Kuwajima, K. (1977) J. Mol. Biol. 114, 241-258[Medline] [Order article via Infotrieve]
23. Kuwajima, K., Mitani, M., and Sugai, S. (1989) J. Mol. Biol. 206, 547-561[Medline] [Order article via Infotrieve]
24. Wu, L. C., Peng, Z. Y., and Kim, P. S. (1995) Nat. Struct. Biol. 2, 281-286[Medline] [Order article via Infotrieve]
25. Wu, L. C., and Kim, P. S. (1998) J. Mol. Biol. 280, 175-182[CrossRef][Medline] [Order article via Infotrieve]
26. Song, J., Bai, P., Luo, L., and Peng, Z. Y. (1998) J. Mol. Biol. 280, 167-174[CrossRef][Medline] [Order article via Infotrieve]
27. Bai, P., Luo, L., and Peng, Z. Y. (2000) Biochemistry 39, 372-380[CrossRef][Medline] [Order article via Infotrieve]
28. Demarest, S. J., Fairman, R., and Raleigh, D. P. (1998) J. Mol. Biol. 283, 279-291[CrossRef][Medline] [Order article via Infotrieve]
29. Schulman, B. A., Kim, P. S., Dobson, C. M., and Redfield, C. (1997) Nat. Struct. Biol. 4, 630-634[Medline] [Order article via Infotrieve]
30. Dill, K. A., and Shortle, D. (1991) Annu. Rev. Biochem. 60, 795-825[CrossRef][Medline] [Order article via Infotrieve]
31. Tanford, C. (1968) Adv. Protein Chem. 23, 121-282[Medline] [Order article via Infotrieve]
32. Demarest, S. J., Boice, J. A., Fairman, R., and Raleigh, D. P. (1999) J. Mol. Biol. 294, 213-211[CrossRef][Medline] [Order article via Infotrieve]
33. Uversky, V. N., Winter, S., and Lober, G. (1996) Biophys. Chem. 60, 79-88[CrossRef][Medline] [Order article via Infotrieve]
34. Klefhaber, T., Labhardt, A. M., and Baldwin, R. L. (1995) Nature 375, 513-515[CrossRef][Medline] [Order article via Infotrieve]
35. Ikeguchi, M., Fujino, M., Kato, M., Kuwajima, K., and Sugai, S. (1998) Protein Sci. 7, 1564-1574[Abstract/Free Full Text]
36. Ikeguchi, M., Sugai, S., Fujino, M., Sugawara, T., and Kuwajima, K. (1992) Biochemistry 31, 12695-12700[Medline] [Order article via Infotrieve]
37. Wu, L. C., Schulman, B. A., Peng, Z. Y., and Kim, P. S. (1996) Biochemistry 35, 859-863[CrossRef][Medline] [Order article via Infotrieve]
38. Polverino, de Laureto, P., De Filippis, V., Di Bello, M., Zambonin, M., and Fontana, A. (1995) Biochemistry 34, 12596-12604[Medline] [Order article via Infotrieve]
39. Polverino, de Laureto, P., Scaramella, E., Frigo, M., Wondrich, F. G., De Filippis, V., Zambonin, M., and Fontana, A. (1999) Protein Sci. 8, 2290-2303[Abstract]
40. Gast, K., Zirwer, D., Muller-Frohne, M., and Damaschun, G. (1998) Protein Sci. 7, 2004-2011[Abstract/Free Full Text]
41. Kataoka, M., Kuwajima, K., Tokunaga, F., and Goto, Y. (1997) Protein Sci. 6, 422-430[Abstract/Free Full Text]
42. Griko, Y. V. (1999) J. Protein Chem. 18, 361-369[Medline] [Order article via Infotrieve]
43. Chang, J.-Y. (1999) J. Biol. Chem. 274, 123-128[Abstract/Free Full Text]
44. Chang, J.-Y., Maerki, W., and Lai, P. H. (1999) Protein Sci. 8, 1463-1468[Abstract]
45. Bulychev, A., and Chang, J.-Y. (1999) J. Prot. Chem. 18, 771-777[Medline] [Order article via Infotrieve]
46. Chang, J.-Y., Li, L., Canals, F., and Aviles, F. X. (2000) J. Biol. Chem. 275, 14205-14211[Abstract/Free Full Text]
47. Tanford, C., Kawahara, K., Lapanje, S., Hooker, T. M., Jr., Zarlengo, M. H., Salahuddin, A., Aune, K. C., and Takagi, T. (1967) J. Am. Chem. Soc. 89, 5023-5029[Medline] [Order article via Infotrieve]
48. Kuwajima, K., Ogawa, Y., and Sugai, S. (1979) Biochemistry 18, 878-882[Medline] [Order article via Infotrieve]
49. Pace, C. N. (1986) Methods Enzymol. 131, 266-280[Medline] [Order article via Infotrieve]
50. Shortle, D., and Meeker, A. K. (1989) Biochemistry 28, 936-944[Medline] [Order article via Infotrieve]
51. Goto, Y., Calciano, L. J., and Fink, A. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 573-577[Abstract]
52. Chang, J.-Y. (1999) Anal. Biochem. 268, 147-150[CrossRef][Medline] [Order article via Infotrieve]
53. Chang, J.-Y., and Ballatore, A. (2000) FEBS Lett. 473, 183-187[CrossRef][Medline] [Order article via Infotrieve]
54. Permyakov, E. A., and Berliner, L. J. (2000) FEBS Lett. 473, 269-274[CrossRef][Medline] [Order article via Infotrieve]
55. Chandra, N., Brew, K., and Acharya, K. R. (1998) Biochemistry 37, 4767-4772[CrossRef][Medline] [Order article via Infotrieve]


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