Author ORCID Identifier
Document Type
Article
Publication Date
12-23-2011
Abstract
Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.
Keywords
diabetes, Mass Spectrometry (MS), molecular modeling, protein structure, radicals, footprinting, proinsulin
Language
43716
Publication Title
Journal of Biological Chemistry
Grant
R01DK040949
Rights
© 2011 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology. This is an Open Access work distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0/) You are free to copy, distribute and transmit the work, provided the original author and source are credited.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Janna G. Kiselar, Manish Datt, Mark R. Chance, Michael A. Weiss, Structural Analysis of Proinsulin Hexamer Assembly by Hydroxyl Radical Footprinting and Computational Modeling*, Journal of Biological Chemistry, Volume 286, Issue 51, 2011, Pages 43710-43716, ISSN 0021-9258, https://doi.org/10.1074/jbc.M111.297853.
Manuscript Version
Final Publisher Version