STRUCTURE AND DYNAMICS OF A PROTEIN ASSEMBLY - H-1-NMR STUDIES OF THE36 KDA R(6) INSULIN HEXAMER

The structure and dynamics of the R(6) human insulin hexamer are inves tigated by two- and three-dimensional homonuclear H-1-NMR spectroscopy The R(6) hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of rec eptor recognition. Despite the large size of the assembly (36 kDa), it s extreme thermal stability permits high-resolution spectra to be obse rved at 55 degrees C. Each spin system is represented uniquely, implyi ng either 6-fold symmetry or fast exchange among allowed protomeric co nformations. Dramatic changes in chemical shifts and long-range nuclea r Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and de monstrates native secondary structure with distinctive R-state N-termi nal extension of the B-chain alpha-helix (residues B1 to B19). The dis tance-geometry structure of an R-state promoter is similar to those of R(6) crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also sim ilar. Although the hexamer is well-ordered in solution, binding of phe nol to an internal cavity occurs within milliseconds, implying the exi stence of ''gatekeeper'' residues whose flexibility provides a portal of entry and release. Changes in H-1-NMR chemical shifts on hexamer as sembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the studies o f insulin analogues (such as ''designed'' insulins of therapeutic inte rest) under conditions of clinical formulation and for the investigati on of the effects of protein assembly on the dynamics of individual pr otomers. (C) 1996 Academic Press Limited