Conformation of 60-residue peptide fragment from N-terminal of porcine kidney fructose 1,6-bisphosphatase |
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Authors: | YANGWeiwenZHAO FukunXU Genjun |
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Institution: | (1) Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 200031 Shanghai, China;(2) State Key Laboratory of Macromolecules, 100101 Beijing, China |
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Abstract: | Limited digestion of fructose 1, 6-bisphosphatase with subtilisin produces an S-peptide with an about 60-residue peptide fragment
that is non-covalently associated with the enzyme. The 60-residue peptide fragment consists of the most part of allosteric
site for AMP binding. It could be separated from S-protein by gel filtration with a Sephadex G-75 column equilibrated with
9% formic acid. According to X-ray diffraction results the S-peptide consists of two α-helices without β-strand and the α-helix
content is about 60% in the 60-residue-peptide fragment. When the enzyme is subjected to limited proteolysis with subtilisin,
the secondary structure of the enzyme does not show a detectable change in CD spectrum. The CD spectra of the isolated S-peptide
were measured under different concentrations. In the absence of GuHCl, S-peptide had 30% α-helix and 38.5% turn-like structure
but had no β-strand, suggesting that the N-terminal 60-residue fragment, which is synthesized initially by ribosome, would
form a conformation spontaneously similar to that of the isolated 60-residue-peptide, i.e. about 30% α-helix and 30% turn-like
structure. As the elongation of the peptide chain of the enzyme proceeds, the newly synthesized segment or the final entire
enzyme, in turn, affects the conformation of prior peptide segment and adjusts its conformation to the final native state.
The content of α-helix did not increase as perturbing the conformation of S-peptide by adding ethanol, cyclohexane or a small
amount of SDS. On the contrary, the ordered structure was slightly decreased, indicating that the difference of conformations
of S-peptide in the isolated form and in the associated protein was not an artifact produced by isolation process.
Project supported in part by the National Natural Science Foundation of China and the Climbing Project of the State Science
and Technology Commission of China. |
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Keywords: | fructose 1 6-bisphosphatase allosteric site secondary structure the folding of newly synthesized peptide conformation adjustment |
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