TY - JOUR
T1 - Thermal cycling aids folding of a recombinant human β-casein with four extra n-terminal amino acid residues
AU - Hu, Y.
AU - Sood, S. M.
AU - Slattery, C. W.
N1 - Author(s): Hu, Y; Sood, SM; Slattery, CW | Abstract: Due to the limited secondary structure, it is believed that the caseins of milk, particularly the beta-caseins (beta-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues.
PY - 2000/11/15
Y1 - 2000/11/15
N2 - Due to the limited secondary structure, it is believed that the caseins of milk, particularly the β-caseins (β-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues. However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into flied tertiary structures. A nonphosphorylated recombinant human β-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism. In 3.3 M urea or at 4°C, the protein was monomeric, as expected. Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native β-CN but with a somewhat different interaction pattern. However, returning the protein to its monomeric state by reequilibration at 4°C followed again by increasing T caused a shift in the pattern. Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed. The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant. The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions. This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland. Further study of a recombinant form with the native amino acid sequence is needed. (C) 2000 Academic Press.
AB - Due to the limited secondary structure, it is believed that the caseins of milk, particularly the β-caseins (β-CN), may be in a mostly random-coil conformation or in various structures that result from random association of hydrophobic residues. However, the self-association of the human proteins with increasing temperature (T) and in the presence of Ca2+ is reproducible, implying that they normally fold into flied tertiary structures. A nonphosphorylated recombinant human β-CN with four extra amino acids at the N-terminus (GSHM-) was prepared and studied by laser light scattering, analytical ultracentrifugation, fluorescence spectroscopy, turbidity, and circular dichroism. In 3.3 M urea or at 4°C, the protein was monomeric, as expected. Increasing T both without and with the addition of Ca2+ ions caused self-association as it does for the nonphosphorylated native β-CN but with a somewhat different interaction pattern. However, returning the protein to its monomeric state by reequilibration at 4°C followed again by increasing T caused a shift in the pattern. Such thermal cycling eventually caused the protein to equilibrate to a particular conformation where no more change could be observed. The resulting interaction pattern was similar to that of the native protein but differed particularly in that there was more extensive self-association for the recombinant mutant. The equilibration to a stable conformation was more rapid in the presence of Ca2+ ions. This suggests that the native protein normally folds into a particular conformation which may be aided by Ca2+ in the mammary gland. Further study of a recombinant form with the native amino acid sequence is needed. (C) 2000 Academic Press.
KW - Analytical ultracentrifugation
KW - Circular dichroism
KW - Fluorescence spectroscopy
KW - Human β-casein
KW - Laser light scattering
KW - Protein folding
KW - Thermal cycling
KW - Turbidity
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U2 - 10.1006/abbi.2000.2063
DO - 10.1006/abbi.2000.2063
M3 - Article
C2 - 11185556
SN - 0003-9861
VL - 383
SP - 215
EP - 224
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -