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Title: Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

Abstract

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives,more » and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.« less

Authors:
; ; ; ; ; ;  [1];  [2];  [2];  [2]
  1. (Antibody Solutions)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGNUNIVERSITYINDUSTRY
OSTI Identifier:
1051107
Resource Type:
Journal Article
Journal Name:
Proc. Natl. Acad. Sci. USA
Additional Journal Information:
Journal Volume: 109; Journal Issue: (37) ; 09, 2012; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AFFINITY; CRYSTALLIZATION; CRYSTALLOGRAPHY; DESIGN; GROWTH FACTORS; MIRRORS; MIXTURES; PLASMONS; PROTEINS; RESOLUTION; RESONANCE; SCREENS; SPECIFICITY; STOICHIOMETRY; SURFACE AREA; SYNTHESIS

Citation Formats

Mandal, Kalyaneswar, Uppalapati, Maruti, Ault-Riché, Dana, Kenney, John, Lowitz, Joshua, Sidhu, Sachdev S., Kent, Stephen B.H., Toronto), Reflexion), and UC). Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography. United States: N. p., 2012. Web. doi:10.1073/pnas.1210483109.
Mandal, Kalyaneswar, Uppalapati, Maruti, Ault-Riché, Dana, Kenney, John, Lowitz, Joshua, Sidhu, Sachdev S., Kent, Stephen B.H., Toronto), Reflexion), & UC). Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography. United States. doi:10.1073/pnas.1210483109.
Mandal, Kalyaneswar, Uppalapati, Maruti, Ault-Riché, Dana, Kenney, John, Lowitz, Joshua, Sidhu, Sachdev S., Kent, Stephen B.H., Toronto), Reflexion), and UC). Tue . "Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography". United States. doi:10.1073/pnas.1210483109.
@article{osti_1051107,
title = {Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography},
author = {Mandal, Kalyaneswar and Uppalapati, Maruti and Ault-Riché, Dana and Kenney, John and Lowitz, Joshua and Sidhu, Sachdev S. and Kent, Stephen B.H. and Toronto) and Reflexion) and UC)},
abstractNote = {Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.},
doi = {10.1073/pnas.1210483109},
journal = {Proc. Natl. Acad. Sci. USA},
issn = {0027-8424},
number = (37) ; 09, 2012,
volume = 109,
place = {United States},
year = {2012},
month = {10}
}