Spectroscopic characterization of the growing polypeptide chain of the barley chymotrypsin inhibitor-2.
A series of N-terminal fragments of increasing size covering the complete sequence of chymotrypsin inhibitor-2 were previously produced using a combination of peptide synthesis and chemical cleavage at engineered methionine residues. Circular dichroism (CD) and NMR spectroscopic studies of the fragments are presented, as well as the folding characterization of the methionine mutants. The far-uv circular dichroism spectra of the small fragments correspond to disordered structures with some weak interactions indicated by temperature effects on certain spectral bands. There is evidence of a formation of a beta-turn around residues 24-26 in fragment CI-2(1-28), not observable on the NMR time scale. The intermediate fragment (1-50) is disordered at low temperature but more ordered when the temperature is increased. The 1D NMR spectra of the large fragments show an increased chemical shift dispersion in the amide hydrogen region, which indicates that tertiary interactions appear in fragment (1-53) together with a major secondary structure change, as shown by the concomitant change in the CD spectra. A key structural transition occurs on addition of residues Phe-50 and Val-51 and fragment (1-60) is largely folded. A second important structural transition in the elongation of the polypeptide occurs on addition of residue Val-63, where a compact fully folded fragment (1-63) is formed albeit with largely reduced stability. Near-uv circular dichroism indicates that the environment of the Trp-5 is fully recovered only in (1-63), probing the correct side-chain packing.
MRC Unit for Protein Function and Design and Cambridge Centre for Protein Engineering, Department of Chemistry, University of Cambridge, United Kingdom.
G
G
de Prat Gay
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D001467:Hordeum; D009682:Magnetic Resonance Spectroscopy; D008961:Models, Structural; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*
D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; C093336:chymotrypsin inhibitor 2; D002918:Chymotrypsin