Solution structure of a designed four-alpha-helix bundle maquette scaffold

The solution structure of a de novo designed disulfide-bridged two-alpha-he lix peptide that self-assembles to form a 2-fold symmetric four-alpha-helix bundle protein (alpha'-SS-alpha')(2) has been solved by NMR spectroscopy. The 33-residue peptide, (alpha'-SH), that is the basic building block of th e bundle has been recombinantly expressed. The three-dimensional structure of the asymmetric unit of the bundle has been determined using interproton distance restraints derived from the nuclear Overhauser effect (NOE), coval ent torsion angle restraints derived from three bond scalar coupling consta nts, and longer range angular restraints derived from residual dipolar coup lings. The covalent alpha'-SS-alpha' unit forms a pair of parallel alpha-he lices that use heptad a-, d-, e-, and g-side chains to form a hydrophobic c ore extending the length of the molecule. The distribution of polar and non polar side chains on the surface of alpha'-SS-alpha' structure is asymmetri c. The hydrophilic face is comprised of glutamate and lysine side chains, w hile the opposite face is comprised of leucine, isoleucine, phenylalanine, tryptophan, and neutral histidine side chains. Equilibrium sedimentation an alysis, size-exclusion chromatography, pulsed field gradient translation di ffusion measurements, and a rotational correlation time derived from N-15 N MR relaxation studies all indicate that the covalent alpha'-SS-alpha' unit forms a noncovalent dimer, (alpha'-SS-alpha')(2), in solution. The structur e confirms many expected design features and illuminates an apparent dichot omy of structure where the helical interface of the disulfide bridged two-a lpha-helix peptide appears nativelike while the adjacent, noncovalent inter face shows non-nativelike behavior. Available evidence indicates the four a lpha-helix bundle can adopt either an anti or syn topology. The structure i s discussed with respect to the potential origins of conformational specifi city and nativelike protein structure.