Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.
Crystals of a fragment of human fibronectin encompassing the 7th through the RGD-containing 10th type III repeats (FN7-10) have been produced with protein expressed in E. coli. The crystals are monoclinic with one molecule in the asymmetric unit and diffract to beyond 2.0 A Bragg spacings. A mutant FN7-10 was produced in which three methionines, in addition to the single native methionine already present, have been introduced by site-directed mutagenesis. Diffraction-quality crystals of this mutant protein have been grown in which methionine was replaced with selenomethionine. The introduction of methionine by site-directed mutagenesis to allow phasing from selenomethionyl-substituted crystals is shown to be feasible by this example and is proposed as a general approach to solving the crystallographic phase problem. Strategies for selecting propitious sites for methionine mutations are discussed.
Duke Scholars
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- Selenomethionine
- Protein Structure, Tertiary
- Peptide Fragments
- Mutagenesis, Site-Directed
- Molecular Sequence Data
- Methionine
- Humans
- Fibronectins
- Crystallography, X-Ray
- Crystallization
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Selenomethionine
- Protein Structure, Tertiary
- Peptide Fragments
- Mutagenesis, Site-Directed
- Molecular Sequence Data
- Methionine
- Humans
- Fibronectins
- Crystallography, X-Ray
- Crystallization