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 p K a 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 ~6% of helical structure, calculated for a 36 residue α-helical peptide. This corresponds to ~20% of that expected for an 11-residue α-helical region. In trifluoroethanol, c.d. measurements indicate that ~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 α-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 α-helix onto the β-sheet.