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
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Abstract |
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Keywords: chimeric protein/domain exchange/molten globule state/protein folding
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Introduction |
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In this study, we constructed a chimeric -LA consisting of the
-domain of human
-LA and the ß-domain of bovine
-LA. First, we indicate the difference in the stability of the molten globule state between recombinant human and bovine
-LA. Then, the factors determining the stability of the molten globule state of
-LA are discussed based on thermodynamic and quantitative investigations of the stability of the constructed chimeric protein's molten globule state.
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Materials and methods |
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Human and bovine -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, 1994
). Refolded proteins were checked by reversed phase HPLC (Uchiyama et al., 1995
). Recombinant proteins showed essentially the same far- and near-UV circular dichroism (CD) spectra and the same biological activity (Fitzgerald et al., 1970
). 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
-domain of bovine
-LA and the ß-domain of human
-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 TrisHCl 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 M1cm1, for authentic and recombinant bovine -LA. An extinction coefficient of 25 900 M1cm1 was used for recombinant human
-LA and chimeric
-LA. Apo proteins were dissolved in 20 mM TrisHCl buffer (pH 8.0) containing various concentrations of guanidine hydrochloride (GuHCl) and 1 mM ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA).
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Results |
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Figure 1 shows amino acid sequences of human, bovine and constructed chimeric
-LAs. Recombinant proteins had an additional Met residue at the N-terminus. We measured the wavelengths of the recombinant proteins at maximum emission,
max, in the presence or absence of Ca2+. The intrinsic fluorescence reflects the environment of the aromatic residues. The parameters
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
-LA and consistent with those of a previous study (Ewbank and Creighton, 1993
).
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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., 1995). 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
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where [] represents the observed ellipticity under given conditions, and [
]MG. and [
]U are ellipticity values at 222 nm in the molten globule and unfolded state, respectively.
Figure 2 shows normalized unfolding curves of recombinant human, bovine and chimeric
-LAs. Gibbs free energy change of unfolding,
G
, for all proteins were estimated from Figure 2
. As is often assumed for the unfolding transition, we assumed that
GU varied linearly with the GuHCl concentration, C; this can be depicted as follows
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where 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
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where R is the gas constant and T is the absolute temperature. Obtained parameters are listed in Table I. The Gibbs free energy change (
G
) and the midpoint (CM) of GuHCl-induced unfolding of recombinant bovine
-LA were smaller than those of recombinant human and chimeric
-LA; and those parameters of the chimeric
-LA (
G
) CM) were almost the same as those of recombinant human
-LA. Values of m for all proteins were almost identical. Furthermore, the stability of the molten globule state of recombinant bovine
-LA was identical to that of authentic
-LA (Figure 3
).
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Discussion |
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It is noteworthy that the stability of the molten globule state of the chimeric protein was found to be equivalent to that of human -LA. These findings suggest that the stability of the molten globule state of
-LA is defined solely by the
-domain. This is consistent with results from previous studies. Recently, Kim and co-workers demonstrated that the molten globule form of
-LA had bipartite structure, the
-domain favoring the native backbone topology and the ß-domain being largely unstructured, by using excellent model proteins which were isolated
-domains (Peng and Kim, 1994
) or two mutants containing only two disulfide bonds in either domain (Wu et al., 1995
). Although these model proteins could not form a native structure, this chimeric protein was able to form a native structure identical to authentic
-LAs. Thus, the characteristic of the molten globule state of this chimeric protein would be close to that of intact
-LAs. This result, which was derived by the thermodynamic analysis with the use of the chimeric protein, indicated that the
-domain was the sole determinant of the stability of the molten globule state of
-LA. Moreover, this demonstrates that the
-domain alone is structured in the molten globule state.
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Notes |
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References |
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Received August 6, 1999; revised October 14, 1999; accepted October 18, 1999.