Simon,
This is not an unusual situation with sulfate, and, yes, sulfate
often occupies phosphate binding sites (particularly when you're work
with 1-3 molar concentration of ammonium sulfate). The simplest way
to remove sulfate from the crystal is to transfer to high
concentrations of citrate. From my own graduate work on GPDH in the
1970's, we just transferred the crystals from 3.0 M AS to 1.44 M Na
citrate. While the crystals weren't stable in citrate for long
periods of time (days), that is not an issue today with
cryocrystallography and flash-freezing. You might even try formate
or malonate, which can have a modest cyroprotectant behavior.
Another, and perhaps more relevant, method is the one devised by Bill
Ray in the late 1980's for phosphoglucomutase (PGM). Apo-PGM was
crystallized in high AS, which caused the same problems as you are
experiencing. Ever the perfectionist, Bill devised a systematic
method to transfer PGM crystals from AS to a PEG solution that
allowed the formation of enzyme complexes (Biochemistry 30, 6866,
1991). Bill also looked at glycine as a replacement as well. So
there are a number of ways to "desalt" crystals without resorting to
crosslinking, while preserving good diffraction characteristics.
Regards,
Michael Garavito
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
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On May 24, 2007, at 2:25 PM, Yue Li wrote:
Hi all,
I have crystals of the apo enzyme growing from 1.7M ammonium sulfate
condition. After soaking with 10mM ligand (substrate analog) which
has a
phosphate group, the ligand did not enter the active site of the
enzyme
because of the competition of sulfate ion and phosphate group. So I am
wondering if anyone knows how to get rid of ammonium sulfate from
crystal before soaking it with the ligand solution? Thanks a lot.
Simon
