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Title: Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions

Abstract

A topic of current interest is engineering surface mutations in order to improve the success rate of protein crystallization. This report explores the possibility of using metal-ion-mediated crystal-packing interactions to facilitate rational design. Escherichia coli apo acyl carrier protein was chosen as a test case because of its high content of negatively charged carboxylates suitable for metal binding with moderate affinity. The protein was successfully crystallized in the presence of zinc ions. The crystal structure was determined to 1.1 {angstrom} resolution with MAD phasing using anomalous signals from the co-crystallized Zn{sup 2+} ions. The case study suggested an integrated strategy for crystallization and structure solution of proteins via engineering surface Asp and Glu mutants, crystallizing them in the presence of metal ions such as Zn{sup 2+} and solving the structures using anomalous signals.

Authors:
;  [1]
  1. (GSK)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1008711
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallogr. D; Journal Volume: 60; Journal Issue: (9) ; 05, 2004
Country of Publication:
United States
Language:
ENGLISH
Subject:
36 MATERIALS SCIENCE; AFFINITY; CRYSTAL STRUCTURE; CRYSTALLIZATION; DESIGN; ENGINEERS; ESCHERICHIA COLI; MUTANTS; MUTATIONS; PROTEINS; RESOLUTION; ZINC IONS

Citation Formats

Qiu, Xiayang, and Janson, Cheryl A. Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions. United States: N. p., 2010. Web.
Qiu, Xiayang, & Janson, Cheryl A. Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions. United States.
Qiu, Xiayang, and Janson, Cheryl A. Tue . "Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions". United States. doi:.
@article{osti_1008711,
title = {Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions},
author = {Qiu, Xiayang and Janson, Cheryl A.},
abstractNote = {A topic of current interest is engineering surface mutations in order to improve the success rate of protein crystallization. This report explores the possibility of using metal-ion-mediated crystal-packing interactions to facilitate rational design. Escherichia coli apo acyl carrier protein was chosen as a test case because of its high content of negatively charged carboxylates suitable for metal binding with moderate affinity. The protein was successfully crystallized in the presence of zinc ions. The crystal structure was determined to 1.1 {angstrom} resolution with MAD phasing using anomalous signals from the co-crystallized Zn{sup 2+} ions. The case study suggested an integrated strategy for crystallization and structure solution of proteins via engineering surface Asp and Glu mutants, crystallizing them in the presence of metal ions such as Zn{sup 2+} and solving the structures using anomalous signals.},
doi = {},
journal = {Acta Crystallogr. D},
number = (9) ; 05, 2004,
volume = 60,
place = {United States},
year = {Tue Nov 16 00:00:00 EST 2010},
month = {Tue Nov 16 00:00:00 EST 2010}
}
  • The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05 and 4.10 Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determiningmore » the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD = 62 ± 13 nM, followed by the binding of two more equivalents of holo-ACPP with KD = 1.2 ± 0.2 μM. Cooperativity was not observed for apo-ACPP which bound with KD = 2.4 ± 0.1 μM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.« less
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  • FabG from A. aeolicus, a putative component of fatty-acid synthase II, has been overexpressed, purified and crystallized. Diffraction data have been collected to 1.8 Å resolution. The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 Å and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative β-ketoacyl-acyl carrier protein reductase. Structure–function studies of this protein are being performed as partmore » of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.« less