期刊
JOURNAL OF BIOMOLECULAR NMR
卷 16, 期 2, 页码 93-108出版社
KLUWER ACADEMIC PUBL
DOI: 10.1023/A:1008323819099
关键词
ambiguous distance restraints; insulin hexamer; principal component analysis; solution structure; symmetric oligomers
Crystallographic and NMR studies of insulin have revealed a highly flexible molecule with a range of different aggregation and structural states; the importance of these states for the function of the hormone is still unclear. To address this question, we have studied the solution structure of the insulin R-6 symmetric hexamer using NMR spectroscopy. Structure determination of symmetric oligomers by NMR is complicated due to `symmetry ambiguity' between intra- and intermonomer NOEs, and between different classes of intermonomer NOEs. Hence, to date, only two symmetric tetramers and one symmetric pentamer (VTB, B subunit of verotoxin) have been solved by NMR; there has been no other symmetric hexamer or higher-order oligomer. Recently, we reported a solution structure for R-6 insulin hexamer. However, in that study, a crystal structure was used as a reference to resolve ambiguities caused by the threefold symmetry; the same method was used in solving VTB. Here, we have successfully recalculated R-6 insulin using the symmetry-ADR method, a computational strategy in which ambiguities are resolved using the NMR data alone. Thus the obtained structure is a refinement of the previous R-6 solution structure. Correlated motions in the final structural ensemble were analysed using a recently developed principal component method; this suggests the presence of two major conformational substates. The study demonstrates that the solution structure of higher-order symmetric oligomers can be determined unambiguously from NMR data alone, using the symmetry-ADR method. This success bodes well for future NMR studies of higher-order symmetric oligomers. The correlated motions observed in the structural ensemble suggest a new insight into the mechanism of phenol exchange and the T-6 <----> R-6 transition of insulin in solution.
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