Parallel β-sheet secondary structure is stabilized and terminated by interstrand disulfide cross-linking

Abstract

Disulfide bonds between Cys residues in adjacent strands of parallel β-sheets are rare among proteins, which suggests that parallel β-sheet structure is not stabilized by such disulfide cross-links. We report experimental results that show, surprisingly, that an interstrand disulfide bond can stabilize parallel β-sheets formed by an autonomously folding peptide in aqueous solution. NMR analysis reveals that parallel β-sheet structure is terminated beyond the disulfide bond, which causes deviation from the extended backbone conformation at one of the Cys residues.

Figure 1

Disulfide bonds between Cys in adjacent strands of (a) anti-parallel β-sheet, in non-hydrogen bond positions, and (b) parallel β-sheet.

Figure 2

Chemical structure of peptide 1-SH. Cysteine residues are shown in red.

Figure 3

NMR-based structural analysis of 1-SS based on data obtained for 2.5 mM peptide in 100 mM deuterioacetate, pH=3.8, 4°C: (a, b) two views of the overlay of the 10 structures with lowest calculated energies from NOE-restrained molecular dynamics simulations; (c, d) two views of the average minimized structure. RMSD among backbone atoms is 1.1 ± 0.4 Å.

Figure 4

¹H NMR chemical shift data for protons attached to C_α (δC_αH) of indicator residues in the unfolded reference peptide (UF₁), 1-SH, 1-SS and the folded reference peptide (FF₁).

Figure 5

Chemical structure of peptide 2-SH. Cysteine residues are shown in red. Indicator residues are denoted by arrows.

Figure 6

(a) ¹H NMR chemical shift data for protons attached to C_α (δC_αH) of indicator residues in the unfolded reference peptide (UF₂), 2-SH, 2-SS and the folded reference peptide (FF₂). (b) Percent folding calculated for 2-SH and 2-SS based on data obtained for 2.5 mM peptide in 100 mM deuterioacetate, pH 3.8, 4°C.