Dominant neg. mutants
ad5...@wayne.edu
> I know i have to mutate the lysin residue which is the ATP binding site
> in the catalytic domain. But my question is how can we find this particular
> lysin residue in the catalytic domain? If somebody has done any x-ray
> studies and these are puiblished could you please send me the info of these
> papers, if anyone of you know??
Regarding MLCK.. There is an X-ray structure of the related twitchin
kinase by the Kemp group, available as Brookhaven PDB file 1KOA. I think
the LYS residue (FMSA-binding) you want is residue 82 in the twitchin
fragment they studied, 17 residues downstream from the third G in the
GLY-rich loop. This would be residue 328 in rat MLCK (tglklaaKvi), for
example.
Regarding CaMK, a structure was published by John Kuriyan's group in Cell
84, 875 (1996).
-------------------------------------------------------------------------------
Anton Scott Goustin, Ph.D. (*)
Center for Molecular Medicine and Genetics
Wayne State University *
5047 Gullen Mall, Room 5167 * *
Detroit, Michigan 48202-3917 USA *
TELEPHONE 313-993-7688 *
FAX 313-577-6200 * UMa
E-mail a...@cmb.biosci.wayne.edu *
WWW http://cmmg.biosci.wayne.edu/asg/asg-home.html
------------------------------------------------------------------------------
"IT IS KNOWN THAT NATURE WORKS CONSTANTLY WITH THE SAME MATERIALS. SHE
IS INGENIOUS TO VARY ONLY THE FORMS." --E. Geoffroy Saint-Hilaire, 1807
==============================================================================
Ananda K. Seneviratne, Ph.D.
I am trying to clone dominant negative mutants of two kinases and a
phosphatase (PP2B, Calcineurin A) which are CaM dependent enzymes.
1. Ca2+-CaM depenedent protein kinase II (CaMKII)
2. Myosin light chain kinase (MLCK)
I know i have to mutate the lysin residue which is the ATP binding site
in the catalytic domain. But my question is how can we find this particular
lysin residue in the catalytic domain? If somebody has done any x-ray
studies and these are puiblished could you please send me the info of these
papers, if anyone of you know??
3. How can i create the same mutant in Calcineurin (a phosphatase)?
Apparecaite your help!
Thanks!
Ananda Seneviratne
Ananda K. Seneviratne, Ph.D.
Department of Biological Sciences,
Wayne State University
Detroit, MI 48202.
(313)577-2665 (Lab)
(313)832-2123 (Home)
Fax:(313)577-6891