2ND-GENERATION OCTARELLINS - 2 NEW DE-NOVO (BETA ALPHA)(8) POLYPEPTIDES DESIGNED FOR INVESTIGATING THE INFLUENCE OF BETA-RESIDUE PACKING ONTHE ALPHA/BETA-BARREL STRUCTURE STABILITY/

The sequence of octarellin I, the first de novo (beta/alpha)(8) polype ptide, was revised according to several criteria, among others the sym metry of the sequence, beta-residue volume and hydrophobicity, and cha rge distribution, These considerations and the overall conclusions dra wn from the first design led to two new sequences, corresponding to oc tarellins II and III, Octarellin II retains perfect 8-fold symmetry. O ctarellin III has the same sequence as octarellin II, except for the b eta-strands which exhibit a 4-fold symmetry, The two proteins were pro duced in Escherichia coli, Infrared and CD spectral analyses of octare llins II and III reveal a high secondary structure content, Non-denatu ring gel electrophoresis, molecular sieve chromatography and analytica l ultracentrifugation suggest that both of these second-generation art ificial polypeptides exist as a mixture of a monomer and a dimer form, Octarellins II and III are at least 10 times more soluble than octare llin I, Urea-induced unfolding followed by fluorescence emission sugge sts that the tryptophan residues, designed to be buried in the (beta/a lpha)(8), are indeed packed in the hydrophobic core of both proteins, However, octarellin III displays a higher stability towards urea denat uration, indicating that introducing 4-fold symmetry into the beta-bar rel might be important for stability of the overall folding.