Efficient Total Chemical Synthesis of 13C=18O Isotopomers of Human Insulin for Isotope‐Edited FTIR |
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| Authors: | Dr. Balamurugan Dhayalan Dr. Ann Fitzpatrick Dr. Kalyaneswar Mandal Dr. Jonathan Whittaker Prof. Dr. Michael A. Weiss Prof. Dr. Andrei Tokmakoff Prof. Dr. Stephen B. H. Kent |
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| Affiliation: | 1. Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA;2. The James Frank Institute, University of Chicago, Chicago, IL, USA;3. Department of Biochemistry, Case Western Reserve University, Cleveland, OH, USA |
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| Abstract: | Isotope‐edited two‐dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site‐specific incorporation of stable 13C=18O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis—via a key ester insulin intermediate—of 97 % enriched [(1‐13C=18O)PheB24] human insulin: stable‐isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X‐ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1‐13C=18O)PheB24] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red‐shifted amide I carbonyl band peak at 1595 cm?1 resulting from the (1‐13C=18O)PheB24 backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function. |
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| Keywords: | chemical protein synthesis human insulin IR spectroscopy isotopic labeling native chemical ligation |
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