Unknown

Dataset Information

0

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.

SUBMITTER: Dhayalan B 

PROVIDER: S-EPMC5477233 | biostudies-other | 2016 Mar

REPOSITORIES: biostudies-other

altmetric image

Publications

Efficient Total Chemical Synthesis of (13) C=(18) O Isotopomers of Human Insulin for Isotope-Edited FTIR.

Dhayalan Balamurugan B   Fitzpatrick Ann A   Mandal Kalyaneswar K   Whittaker Jonathan J   Weiss Michael A MA   Tokmakoff Andrei A   Kent Stephen B H SB  

Chembiochem : a European journal of chemical biology 20160216 5


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 %  ...[more]

Similar Datasets

| S-EPMC10116525 | biostudies-literature
| S-EPMC4668202 | biostudies-literature
| S-EPMC3152580 | biostudies-literature
| S-EPMC3826463 | biostudies-literature
| S-EPMC3835593 | biostudies-literature
| S-EPMC5810247 | biostudies-literature
| S-EPMC2894265 | biostudies-literature
| S-EPMC9241142 | biostudies-literature
| S-EPMC3757576 | biostudies-literature
| S-EPMC10786670 | biostudies-literature