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Title: Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16

Abstract

The crystallization and crystallographic data analysis of filamin repeats 14–16 are reported. Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14–16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 Å and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 Å, α = β = γ = 90°.

Authors:
 [1];  [2]; ;  [1];  [1];  [2]
  1. Department of Medical Biochemistry and Microbiology, Uppsala University (Sweden)
  2. (Singapore)
Publication Date:
OSTI Identifier:
22360291
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 4; Other Information: PMCID: PMC2330200; PMID: 17401197; PUBLISHER-ID: bo5013; OAI: oai:pubmedcentral.nih.gov:2330200; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
36 MATERIALS SCIENCE; 75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; 60 APPLIED LIFE SCIENCES; AMMONIUM SULFATES; CROSS-LINKING; CRYSTALLIZATION; CRYSTALS; DATA ANALYSIS; ESCHERICHIA COLI; FILAMENTS; MICROTUBULES; RODS; SPACE GROUPS

Citation Formats

Aguda, Adeleke Halilu, E-mail: adelekeha@imcb.a-star.edu.sg, Institute of Molecular and Cell Biology, Sakwe, Amos Malle, Rask, Lars, Robinson, Robert Charles, and Institute of Molecular and Cell Biology. Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107006689.
Aguda, Adeleke Halilu, E-mail: adelekeha@imcb.a-star.edu.sg, Institute of Molecular and Cell Biology, Sakwe, Amos Malle, Rask, Lars, Robinson, Robert Charles, & Institute of Molecular and Cell Biology. Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16. United Kingdom. doi:10.1107/S1744309107006689.
Aguda, Adeleke Halilu, E-mail: adelekeha@imcb.a-star.edu.sg, Institute of Molecular and Cell Biology, Sakwe, Amos Malle, Rask, Lars, Robinson, Robert Charles, and Institute of Molecular and Cell Biology. Sun . "Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16". United Kingdom. doi:10.1107/S1744309107006689.
@article{osti_22360291,
title = {Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14–16},
author = {Aguda, Adeleke Halilu, E-mail: adelekeha@imcb.a-star.edu.sg and Institute of Molecular and Cell Biology and Sakwe, Amos Malle and Rask, Lars and Robinson, Robert Charles and Institute of Molecular and Cell Biology},
abstractNote = {The crystallization and crystallographic data analysis of filamin repeats 14–16 are reported. Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14–16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 Å and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 Å, α = β = γ = 90°.},
doi = {10.1107/S1744309107006689},
journal = {Acta Crystallographica. Section F},
number = Pt 4,
volume = 63,
place = {United Kingdom},
year = {Sun Apr 01 00:00:00 EDT 2007},
month = {Sun Apr 01 00:00:00 EDT 2007}
}
  • A unique feature of cyanobacteria genomes is the abundance of genes that code for hypothetical proteins containing tandem pentapeptide repeats approximately described by the consensus motif A[N/D]LXX. Too date, structures of two pentapeptide repeat proteins (PRPs) have been determined with the tandem pentapeptide repeat sequences observed to adopt a novel right-handed quadrilateral b-helix, or Rfr-fold, in both structures. One structure, Mycobacterium tuberculosis MfpA, is a 183-residue protein that contains 30 consecutive pentapeptide repeats and appears to offer antibiotic resistance by acting as a DNA mimic. The other structure, Cyanothece Rfr32, is a 167-residue protein that contains 21 consecutive pentapeptide repeats.more » The function of Rfr32, like the other 35 hypothetical PRPs identified in the genome of Cyanothece, is unknown. In an effort to understand the role of PRPs in cyanobacteria, and to better characterize the structural properties of Rfr-folds with different amino acid sequences, a second PRP from Cyanothece 51142, Rfr23, has been cloned, expressed, and purified. Selenomethione substituted protein was crystallized by vapor diffusion in hanging drops. MAD diffraction data were collected on these crystals to 2.? Å resolution using synchrotron radiation. The crystals belonged to space group I41 with unit-cell parameters a = b = 106.23 Å, c = 52.40 Å. Analysis of the 172-residue protein sequence suggests that Rfr23 contains 26 pentapeptide repeats interrupted by eight residues near the N-terminus. The electron density map suggests that the pentapeptide repeats adopt a similar right-handed quadrilateral b-helix as observed in the other two PRP structures, however, the eight residue interruption in the string of pentapeptide repeats appears to create a distortion in the Rfr-fold.« less
  • A unique feature of cyanobacteria genomes is the abundance of genes that code for hypothetical proteins containing tandem pentapeptide repeats approximately described by the consensus motif A(N/D)LXX. To date, the structures of two pentapeptide-repeat proteins (PRPs) have been determined, with the tandem pentapeptide-repeat sequences observed to adopt a novel type of right-handed quadrilateral {beta}-helix, or Rfr-fold, in both structures. One structure, Mycobacterium tuberculosis MfpA, is a 183-residue protein that contains 30 consecutive pentapeptide repeats and appears to offer antibiotic resistance by acting as a DNA mimic. The other structure, Cyanothece 51142 Rfr32, is a 167-residue protein that contains 21 consecutivemore » pentapeptide repeats. The function of Rfr32, like the other 35 hypothetical PRPs identified in the genome of Cyanothece, is unknown. In an effort to understand the role of PRPs in cyanobacteria and to better characterize the structural properties of Rfr-folds with different amino-acid sequences, a second PRP from Cyanothece 51142, Rfr23, has been cloned, expressed and purified. Selenomethione-substituted protein was crystallized by vapor diffusion in hanging drops. Nearly complete SAD and native diffraction data sets were collected from these crystals to 2.5 and 2.1 {angstrom} resolution, respectively, using synchrotron radiation. The crystals belonged to space group I4{sub 1}, with unit-cell parameters a = b = 106.61, c = 53.37 {angstrom}, and one molecule per asymmetric unit. Preliminary analysis of the electron-density map from the SAD data shows that Rfr23 contains an Rfr-fold.« less
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