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

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-Th r-Ile-Lys-(D)pro-Gly-Ile-Thr-Phe-Ala-(D)Pro-Ala-Thr-Val-Leu-Phe-Ala-Val-(D) Pro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically posit ioned (D)Pro-Xxx segments nucleate type II' beta -turns, which facilitate h airpin extension. A four-stranded beta -sheet structure is determined in me thanol from 500 MHz H-1 NMR data using a total of 100 observed NOEs, 11 dih edral restraints obtained from vicinal J(C alphaH-NH) values and 10 hydroge n bonding constraints obtained from H/D exchange data. The observed NOEs pr ovide strong evidence for a stable four-stranded sheet and a nonpolar clust er involving Ile(8), Phe(10), Val(15) and Phe(17). Circular dichroism studi es in water-methanol mixtures provide evidence for melting of the P-sheet s tructure at high water concentrations. NMR analysis establishes that the fo ur-stranded sheet in Beta-4 is appreciably populated in 50% (v/v) aqueous m ethanol. In water, the peptide structure is disorganized, although the thre e beta -turn nuclei appear to be maintained.