Protocol

MAP Kinase Signaling Protocols

Volume 661 of the series Methods in Molecular Biology pp 223-237

Date:

Structural Studies of MAP Kinase Cascade Components

  • Elizabeth J. GoldsmithAffiliated withDepartment of Biochemistry, The University of Texas Southwestern Medical Center at Dallas Email author 
  • , Xiaoshan MinAffiliated withDepartment of Biochemistry, The University of Texas Southwestern Medical Center at Dallas
  • , Haixia HeAffiliated withDepartment of Biochemistry, The University of Texas Southwestern Medical Center at Dallas
  • , Tianjun ZhouAffiliated withDepartment of Biochemistry, The University of Texas Southwestern Medical Center at Dallas

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Abstract

MAPK cascade components have been the subject of structural analysis, advancing our understanding of how these enzymes are activated and how they interact. A surprising finding has been that unique inactive conformers are adopted by many of these kinases. These inactive conformers are interesting and often require experimental phases to determine their crystal structures because molecular replacement techniques are not successful. Here, we describe the preparation of MAP2K MEK6 and MAP3K TAO2 substituted with selenomethionine (SeMet) for de novo phasing. TAO2 and SeMet TAO2 were expressed in insect cells.

Key words

MAPK MAP2K MAP3K MEK6 TAO2 Selenomethionine Crystallography Docking ERK2 p38alpha