Synthetic protein design: construction of a four-stranded beta-sheet structure and evaluation of its integrity in...

The characterization of a four-stranded beta-sheet structure in a designed 26-residue peptide Beta-4 is described. The sequence of Beta-4 (Arg-Gly-Thr-Ile-Lys-(D)pro-Gly-Ile-Thr-Phe-Ala-(D)Pro-Ala-Thr-Val-Leu-P he-Ala-Val-(D)Pro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically positioned (D)Pro-Xxx segments nucleate type II' beta-turns, which facilitate hairpin extension. A four-stranded beta-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 JCalphaH-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 beta-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 beta-turn nuclei appear to be maintained.