Study on the packing geometry, stoichiometry, and membrane interaction of three analogs related to a pore-forming small globular protein

A de novo designed pore-forming small globular protein (SGP) with antitumor activity consists of four helices: 3 basic amphipathic helices composed of Leu and Lys surrounding a central hydrophobic helix composed of oligoalani ne. These helices are connected by a beta -turn-forming sequence and two be ta -turn-unfavorable ones (S. Lee, T. Kiyota, T. Kunitake, E. Matsumoto, S. Yamashita, K. Anzai, and G. Sugihara Biochemistry 1997, Vol. 36, pp. 3782- 3791). In the present work, we designed and synthesized three new SGP analo gs in order to study the stoichiometric packing geometry and stability of S GP. The replacement of alanines in the central helix of SGP with leucines ( SGP-L), which make the helix much larger in size and more hydrophobic, resu lted in tin equilibrium of monomeric-trimeric structure. The replacement of some Lys residues by Glu residues in the hydrophilic regions of the amphip athic helices (SGP-E) led to a decrease in helical content and the formatio n of an equilibrium of monomeric-trimeric structure. The alteration of beta -turn regions with Gly residues, which makes these regions flexible (SGP-G ), established tin equilibrium of monomeric-dimeric states in buffer. The h ydrophobic alpha -helix of SGP-L penetrated into the lipid bilayers in a ma nner that stabilized model membranes and biomembranes, whereas the central helices of SGP-G and -E destabilized them by forming channels. SGP and its analogs may be a useful model to study the role of the hydrophobic and hydr ophilic regions in the formation of monomer-oligomer of proteins and to bet ter understand the insertion of membrane targeting proteins into biomembran es. (C) 2001 John Wiley & Sons, Inc.