Synthetic protein design: construction of a four-stranded β-sheet structure and evaluation of its integrity in methanol-water systems

Abstract

The characterization of a four-stranded β-sheet structure in a designed 26-residue peptide Beta-4 is described. The sequence of Beta-4 (Arg-Gly-Thr-Ile-Lys-^Dpro-Gly-Ile-Thr-Phe-Ala-^DPro-Ala-Thr-Val-Leu-Phe-Ala-Val-^DPro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically positioned ^DPro-Xxx segments nucleate type II'β-turns, which facilitate hairpin extension. A four-stranded β-sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH-NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four-stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water-methanol mixtures provide evidence for melting of the β-sheet structure at high water concentrations. NMR analysis establishes that the four-stranded sheet in Beta-4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β-turn nuclei appear to be maintained.