title: Conformation of 60-residue peptide fragment from N-terminal of porcine kidney fructose 1,6-bisphosphatase.
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 alpha-helices without beta-strand and the alpha-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% alpha-helix and 38.5% turn-like structure but had no beta-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% alpha-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 alpha-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.
authors: Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 200031, Shanghai, China.
authors: W
authors: W
authors: Yang
authors: F
authors: F
authors: Zhao
authors: G
authors: G
authors: Xu