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4S-Hydroxylation of insulin at ProB28 accelerates hexamer dissociation and delays fibrillation

Lieblich, Seth A. and Fang, Katharine Y. and Cahn, Jackson K. B. and Rawson, Jeffrey and LeBon, Jeanne and Ku, H. Teresa and Tirrell, David A. (2017) 4S-Hydroxylation of insulin at ProB28 accelerates hexamer dissociation and delays fibrillation. Journal of the American Chemical Society, 139 (25). pp. 8384-8387. ISSN 0002-7863. PMCID PMC5812673. https://resolver.caltech.edu/CaltechAUTHORS:20170612-160413860

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Abstract

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/jacs.7b00794DOIArticle
http://pubs.acs.org/doi/abs/10.1021/jacs.7b00794PublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/jacs.7b00794PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812673PubMed CentralArticle
ORCID:
AuthorORCID
Lieblich, Seth A.0000-0003-2705-5821
Tirrell, David A.0000-0003-3175-4596
Additional Information:© 2017 American Chemical Society. Received: January 24, 2017. Published: June 9, 2017. We thank J. T. Kaiser, P. Nikolovski, S. Russi, S. Virgil, M. Shahgholi, A. Lakshmanan, and the scientific staff of Beamline 12-2 at the Stanford Synchrotron Radiation Laboratory for assistance. We thank W. Glenn, A. Mahdavi, and T. Hoeg-Jensen for discussions. The work was supported by the Novo Nordisk Foundation. Fellowships from Amgen and from the Natural Sciences and Engineering Research Council of Canada (NSERC, PGS-D) provided partial support for S.A.L. and K.Y.F., respectively. J.K.B.C. acknowledges support of the Resnick Sustainability Institute (Caltech). The authors declare the following competing financial interest(s): S.A.L., K.Y.F., and D.A.T are inventors on a related patent application.
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
Novo Nordisk FoundationUNSPECIFIED
AmgenUNSPECIFIED
Natural Sciences and Engineering Research Council of Canada (NSERC)UNSPECIFIED
Resnick Sustainability InstituteUNSPECIFIED
Issue or Number:25
PubMed Central ID:PMC5812673
Record Number:CaltechAUTHORS:20170612-160413860
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170612-160413860
Official Citation:4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation Seth A. Lieblich, Katharine Y. Fang, Jackson K. B. Cahn, Jeffrey Rawson, Jeanne LeBon, H. Teresa Ku, and David A. Tirrell Journal of the American Chemical Society 2017 139 (25), 8384-8387 DOI: 10.1021/jacs.7b00794
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:78131
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Jun 2017 23:15
Last Modified:03 Oct 2019 18:05

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