Abstract: | 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 JCαH‐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. |