title: An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate.
abstract: A fragment of barnase comprising amino acids 1 to 36 (B(1-36)) that encompasses the region containing the two large helices (residues 6-18 and 26-34) of the native protein has been obtained by cleavage of the barnase mutant Val36----Met with cyanogen bromide. The circular dichroism (c.d.) spectrum of B(1-36) in the far ultraviolet indicates that the fragment is only weakly structured in water at neutral pH. The two-dimensional 1H nuclear magnetic resonance spectrum of B(1-36) shows, however, that a fraction of the population does have helical structure, spanning amino acid residues 8 to 18. B(1-36) becomes more helical in 35% trifluoroethanol. This is indicated by the c.d. spectrum and the increase from 6.6 to 7.0 in the pKa of His18, which is known to interact with the dipole of helix 6-18 in native barnase. The helical region of B(1-36) in 35% trifluoroethanol extends to residue 6. It is calculated from extrapolation of a trifluoroethanol titration of the ellipticity at 222 nm that B(1-36) exhibits in water approximately 6% of helical structure, calculated for a 36 residue alpha-helical peptide. This corresponds to approximately 20% of that expected for an 11-residue alpha-helical region. In trifluoroethanol, c.d. measurements indicate that approximately 30% of the 36-residue peptide is helical. It has been shown from extensive studies of the refolding of barnase that there is a folding intermediate that contains residues 8 to 18 in a helical conformation and that residue 6 is mainly unfolded. The experiments on the conformation of B(1-36) show that a small, but significant fraction, of its population in water adopts the conformation of the major alpha-helix during the barnase folding pathway, in the absence of tertiary interactions. Thus, in the folding of native barnase, secondary structure formation can precede the docking of the major alpha-helix onto the beta-sheet.
authors.affiliation: Department of Chemistry, University of Cambridge, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Sancho
authors.forename: J L
authors.initials: JL
authors.lastname: Neira
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014270:Trifluoroethanol / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D014270:Trifluoroethanol; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease