Crystallization of a fragment of human fibronectin: introduction of methionine by site-directed mutagenesis to allow phasing via selenomethionine.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Leahy, DJ; Erickson, HP; Aukhil, I; Joshi, P; Hendrickson, WA

Published Date

  • May 1994

Published In

Volume / Issue

  • 19 / 1

Start / End Page

  • 48 - 54

PubMed ID

  • 8066086

Pubmed Central ID

  • 8066086

International Standard Serial Number (ISSN)

  • 0887-3585

Digital Object Identifier (DOI)

  • 10.1002/prot.340190107

Language

  • eng

Conference Location

  • United States