Structure and dynamics of a protein assembly. 1H-NMR studies of the 36 kDa R6 insulin hexamer.
The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 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 similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly 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 interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers.
Jacoby
E
E
Center on Biomolecular Structure and Function, University of Chicago, IL 60637, USA.
Hua
Q X
QX
Stern
A S
AS
Frank
B H
BH
Weiss
M A
MA
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006358:Hot Temperature; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding
D007328:Insulin