Stability of the molten globule state of a domain-exchanged chimeric protein between human and bovine {alpha}-lactalbumins

Kazuo Masaki4, Ryusuke Masuda, Kenji Takase1, Keiichi Kawano2 and Katsutoshi Nitta3

Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, 1 Department of Biotechnology, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 and 2 Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, 4 Department of Insect Physiology and Behavior, National Institute of Sericultural and Entomological Science, Ministry of Agriculture, Forestry and Fisheries, Oowashi 1-2, Tsukuba, Ibaraki 305-8634, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
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A domain-exchanged chimeric {alpha}-lactalbumin ({alpha}-LA), which consisted of the {alpha}-domain of human {alpha}-LA and the ß-domain of bovine {alpha}-LA, was constructed. Like native {alpha}-LA, the chimeric protein was in a molten globule state in the absence of Ca2+ at neutral pH and low salt concentration. The stability of the molten globule state of the constructed chimeric protein was identical to that of the recombinant human protein and was higher than that of the recombinant bovine protein. The stability of the molten globule state of {alpha}-LA is defined by the stability of the {alpha}-domain.

Keywords: chimeric protein/domain exchange/molten globule state/protein folding


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structure of {alpha}-lactalbumin ({alpha}-LA) consists of a {alpha}-helical domain ({alpha}-domain; residues 1–34 and 86–123) and a ß-sheet domain (ß-domain; residues 35–85); there exists a deep cleft and a Ca2+ binding site between the two domains (Pike et al., 1996Go). It is known that {alpha}-LA is a Ca2+ binding protein (Hiraoka et al., 1980Go) and that the overall structure of {alpha}-LA is very similar to that of the c-type lysozyme, which usually cannot bind Ca2+ (Acharya et al., 1989Go). {alpha}-Lactalbumin is in a molten globule state at low pH, or at low salt concentration and neutral pH in the apo state (Dolgikh et al., 1981Go; Segawa and Sugai, 1983Go). The molten globule state of {alpha}-LA is the most characterized one. The equilibrium molten globule state has been shown to be similar to a kinetic intermediate in the refolding reaction (Ikeguchi et al., 1986Go; Kuwajima, 1989Go; Arai and Kuwajima, 1996Go). NMR studies have indicated that the regions most protected from hydrogen exchange in the molten globule state are the helical regions in the native state (Schulman et al., 1995Go). These findings suggest that helical structures in the molten globule state are almost identical to those in the native state. It has been demonstrated that the molten globule form of {alpha}-LA has a bipartite structure, as determined qualitatively by the analysis of the disulfide rearrangement (Wu et al., 1995Go). On the other hand, the stability of the molten globule state of goat {alpha}-LA has been studied by using mutant proteins (Uchiyama et al., 1995Go) and on the basis of this study it has been suggested that the hydrophobic core in {alpha}-LA plays an important role in stabilizing the molten globule state.

In this study, we constructed a chimeric {alpha}-LA consisting of the {alpha}-domain of human {alpha}-LA and the ß-domain of bovine {alpha}-LA. First, we indicate the difference in the stability of the molten globule state between recombinant human and bovine {alpha}-LA. Then, the factors determining the stability of the molten globule state of {alpha}-LA are discussed based on thermodynamic and quantitative investigations of the stability of the constructed chimeric protein's molten globule state.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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Construction of chimeric {alpha}-LA

Human and bovine {alpha}-LA cDNAs were derived by PCR from QUICK-CloneTM Human Mammary Gland cDNA (Clontech) and Bovine Mammary cDNA Library in the Uni-ZAPTM XR Vector (Stratagene), respectively. The cDNA of the chimeric protein was prepared by the ligation of gel-purified DNA fragments obtained by digestion with FokI and BsrI. All proteins were expressed using the pET 22-b(+) vector (Novagen) as inclusion bodies in Escherichia coli strain BL21 (DE3). The refolding reaction of all proteins was performed as described previously (Peng and Kim, 1994Go). Refolded proteins were checked by reversed phase HPLC (Uchiyama et al., 1995Go). Recombinant proteins showed essentially the same far- and near-UV circular dichroism (CD) spectra and the same biological activity (Fitzgerald et al., 1970Go). The additional Met residue at the N-terminus of the recombinant proteins was confirmed by an analysis of the amino acid sequence. We attempted to construct another chimeric protein, which consisted of the {alpha}-domain of bovine {alpha}-LA and the ß-domain of human {alpha}-LA, but that did not refold.

Intrinsic fluorescence

Intrinsic fluorescence measurements were performed with a fluorescence spectrophotometer 650-60 (Hitachi). Recombinant proteins were dissolved to 1.8 mM in 20 mM Tris–HCl buffer (pH 8.0) in the absence or presence of 1 mM CaCl2. The emission spectra were recorded at 25°C between 300 and 400 nm using excitation at 280 nm.

Circular dichroism measurements

Circular dichroism measurements were performed with a J-725 spectropolarimeter (Jasco). The path length of the optical cuvette was 1 mm for the measurements at 222 nm. The temperature was controlled at 25°C with a bath circulator RTE-110 (NESLAB). Protein concentrations were determined by absorbance at 280 nm using an extinction coefficient, 28 500 M–1cm–1, for authentic and recombinant bovine {alpha}-LA. An extinction coefficient of 25 900 M–1cm–1 was used for recombinant human {alpha}-LA and chimeric {alpha}-LA. Apo proteins were dissolved in 20 mM Tris–HCl buffer (pH 8.0) containing various concentrations of guanidine hydrochloride (GuHCl) and 1 mM ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA).


    Results
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Molten globule state of recombinant human, bovine and chimeric {alpha}-LAs

Figure 1Go shows amino acid sequences of human, bovine and constructed chimeric {alpha}-LAs. Recombinant proteins had an additional Met residue at the N-terminus. We measured the wavelengths of the recombinant proteins at maximum emission, {lambda}max, in the presence or absence of Ca2+. The intrinsic fluorescence reflects the environment of the aromatic residues. The parameters {lambda}max of all the proteins were red-shifted by removing Ca2+, indicating that aromatic residues, especially Trp residues, of apo proteins were in a more hydrophilic environment relative to that in the Ca2+ bound state. These observations are characteristic of the molten globule state of {alpha}-LA and consistent with those of a previous study (Ewbank and Creighton, 1993Go).



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Fig. 1. Amino acid sequence of bovine, human and chimeric {alpha}-LAs. Recombinant proteins have an additional Met residue at the N-terminus. The {alpha}-domain region is underlined. Dots (.) represent amino acids that are identical in the chimeric protein to those in the bovine and human {alpha}-LAs.

 
Furthermore, far- and near-UV CD spectra of apo proteins indicated that, in the absence of Ca2+, all recombinant proteins were in the molten globule state (Figure 2Go). Under this condition (20 mM Tris–HCl and 1 mM EGTA at pH 8.0), it was shown that the authentic protein was also in the molten globule state (Arai and Kuwajima, 1996Go). However, in the presence of Ca2+, far- and near-UV CD spectra of all recombinant proteins, including the chimeric protein, were identical to those of the holo form of the authentic proteins.



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Fig. 2. CD spectra of apo- and holo-forms of recombinant {alpha}-LAs. Thin lines represent CD spectra of the holo-form (20 mM Tris–HCl pH 8.0 and 1 mM CaCl2 at 25°C) and thick lines represent CD spectra of the apo-form (20 mM Tris–HCl pH 8.0 and 1 mM EGTA at 25°C). Solid (), dotted (. . .) and broken (- - -) lines represent CD spectra of recombinant chimeric, human and bovine {alpha}-LAs, respectively.

 
Guanidine hydrochloride-induced unfolding transitions of apo recombinant human, bovine and chimeric {alpha}-LAs

The GuHCl-induced unfolding of all proteins was assumed to be a two-state process according to the previous study of the stability of the molten globule state (Uchiyama et al., 1995Go). The GuHCl-induced unfolding of the apo proteins was monitored by CD ellipticity at 222 nm, which is commonly used as a measure of the extent of secondary structures. The fractional extent of unfolding, fU, was calculated from the ellipticity values using the equation


where [{theta}] represents the observed ellipticity under given conditions, and [{theta}]MG. and [{theta}]U are ellipticity values at 222 nm in the molten globule and unfolded state, respectively.

Figure 2Go shows normalized unfolding curves of recombinant human, bovine and chimeric {alpha}-LAs. Gibbs free energy change of unfolding, {Delta}G, for all proteins were estimated from Figure 2Go. As is often assumed for the unfolding transition, we assumed that {Delta}GU varied linearly with the GuHCl concentration, C; this can be depicted as follows


where {Delta}G is the Gibbs free energy change at 0 M GuHCl, CM is the midpoint value of GuHCl concentration of the unfolding transition, and m indicates the cooperativity parameter of the unfolding. The unfolding curve is expressed by the following equation, which is based on the assumption of a two-state transition



where R is the gas constant and T is the absolute temperature. Obtained parameters are listed in Table IGo. The Gibbs free energy change ({Delta}G) and the midpoint (CM) of GuHCl-induced unfolding of recombinant bovine {alpha}-LA were smaller than those of recombinant human and chimeric {alpha}-LA; and those parameters of the chimeric {alpha}-LA ({Delta}G) CM) were almost the same as those of recombinant human {alpha}-LA. Values of m for all proteins were almost identical. Furthermore, the stability of the molten globule state of recombinant bovine {alpha}-LA was identical to that of authentic {alpha}-LA (Figure 3Go).


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Table I. Guanidine hydrochloride-induced unfolding parameters of apo recombinant {alpha}-lactalbumins
 


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Fig. 3. Guanidine hydrochloride-induced transition of the molten globule to the unfolded state. Equation 1 was used to calculate unfolded fractions (fu) of apo recombinant bovine ({square}), human ({circ}) and chimeric proteins ({blacktriangleup}); the fractions were calculated from the ellipticity values at 222 nm. Dotted lines represent the theoretical curves calculated from Eqn 3 using the parameters shown in Table IGo. Cross symbols (+) indicate the unfolded fractions of authentic bovine apo-protein.

 
On the other hand, ellipticity values of all apo proteins at 270 nm in 6 M GuHCl were identical to those in the absence of GuHCl, in the molten globule state.


    Discussion
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 Abstract
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 Materials and methods
 Results
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The stability of the molten globule state of recombinant bovine {alpha}-LA was lower than that of recombinant human {alpha}-LA (Figure 3Go). Furthermore, the stability of the molten globule state of the constructed chimeric protein was identical to that of human {alpha}-LA. What is responsible for the difference in the stability of the molten globule state between human and bovine {alpha}-LA? Recently, amino acid residues located in the B helix of goat {alpha}-LA were changed into more hydrophobic residues and it was demonstrated that the molten globule state of mutant proteins was stabilized by the substitutions (Uchiyama et al., 1995Go). Thus, the hydrophobic interaction is important for the stabilization of the molten globule state. Furthermore, we assume that the difference in amino acid residues forming the hydrophobic interaction contributes to the difference in the stability of the molten globule state between human and bovine {alpha}-LA. According to an NMR study, two aromatic clusters which existed in the {alpha}-domain of the native protein were not detected in the molten globule state (Alexandrescu et al., 1993Go). However, it has been suggested that some specific native-like packing, which consists of A, B and C-terminal 310 helices [residues 5–11, 23–34 and 115–118, respectively (Pike et al., 1996Go)], exists in the core of the molten globule state (Wu and Kim, 1998Go). In this region, eight amino acids are substituted between human and bovine {alpha}-LA (Figure 1Go). These substitutions result in an increase in hydrophobicity in this region of human {alpha}-LA (Kyte and Doolittle, 1982Go). In the molten globule state, such differences might result in differences in stability. Moreover, the difference in the amino acid residue at position 30 in the B helix might result in a difference in stability of the molten globule state between human and bovine {alpha}-LAs. It has been demonstrated, for example, that the molten globule state of the Ala30Ile mutant of goat {alpha}-LA is more stable than that of wild-type {alpha}-LA. Because the amino acid sequence in this region is identical in both human {alpha}-LA and the constructed chimeric protein, the molten globule state of the chimeric protein should also be more stable than that of bovine {alpha}-LA.

It is noteworthy that the stability of the molten globule state of the chimeric protein was found to be equivalent to that of human {alpha}-LA. These findings suggest that the stability of the molten globule state of {alpha}-LA is defined solely by the {alpha}-domain. This is consistent with results from previous studies. Recently, Kim and co-workers demonstrated that the molten globule form of {alpha}-LA had bipartite structure, the {alpha}-domain favoring the native backbone topology and the ß-domain being largely unstructured, by using excellent model proteins which were isolated {alpha}-domains (Peng and Kim, 1994Go) or two mutants containing only two disulfide bonds in either domain (Wu et al., 1995Go). Although these model proteins could not form a native structure, this chimeric protein was able to form a native structure identical to authentic {alpha}-LAs. Thus, the characteristic of the molten globule state of this chimeric protein would be close to that of intact {alpha}-LAs. This result, which was derived by the thermodynamic analysis with the use of the chimeric protein, indicated that the {alpha}-domain was the sole determinant of the stability of the molten globule state of {alpha}-LA. Moreover, this demonstrates that the {alpha}-domain alone is structured in the molten globule state.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received August 6, 1999; revised October 14, 1999; accepted October 18, 1999.