title: Efficient Total Chemical Synthesis of (13) C=(18) O Isotopomers of Human Insulin for Isotope-Edited FTIR.
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 (13) C=(18) O 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-(13) C=(18) O)Phe(B24) ] 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-(13) C=(18) O)Phe(B24) ] 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-(13) C=(18) O)Phe(B24) 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.
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA.
authors.forename: Balamurugan
authors.initials: B
authors.lastname: Dhayalan
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA.|The James Frank Institute, University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Fitzpatrick
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA.
authors.forename: Kalyaneswar
authors.initials: K
authors.lastname: Mandal
authors.affiliation: Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.affiliation: Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Weiss
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA. tokmakoff@uchicago.edu.|The James Frank Institute, University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA. tokmakoff@uchicago.edu.
authors.forename: Andrei
authors.initials: A
authors.lastname: Tokmakoff
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA. skent@uchicago.edu.
authors.forename: Stephen B H
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D002247:Carbon Isotopes / Q000737:chemistry*; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D007328:Insulin; D010103:Oxygen Isotopes
keywords: IR spectroscopy; chemical protein synthesis; human insulin; isotopic labeling; native chemical ligation