Re: [ccp4bb] DTT sensitive?

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: CCP4 Study Weekend cost increase from Sat. 17 Nov.
From: Charles Ballard c {- dot -} c {- dot -} ballard {- at -} DL {- dot -} AC {- dot -} UK
Date: 2007-11-14

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Subject: Fwd: Re: Protein powder diffraction (fwd)
From: Jonathan Wright wright {- at -} ESRF {- dot -} FR
Date: 2007-11-14

Subject: Re: DTT sensitive?
From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU
Date: 2007-11-14

Joe,

What your are describing is microcrystal formation. The silkiness or
"opalescence" is very typical of microcrystalline showers.
Concerning the effect of DTT, I recall one other case where DTT-
sensitive microcrystals formed: the crystalline insecticidal toxins
produced by B. thuringiensis, which are used as specific
insecticides. Although I can't recall the details, possible
disulfide bridge formation occurred during crystal formation. Jade
Li solved its structure awhile ago (Li, Koni, and Ellar, J. Mol.
Biol. 257, 129–152, 1996) and noted:

"Crystals of the CytB protoxin were grown (Li et al., 1995) by
microdialysis in the presence of 5 mM dithiothreitol (DTT), by
reducing the pH from 8.5 to 7.2 and at the same time reducing the
concentration of a solubilizing agent, either urea from 50 mM to nil,
or ethanolamine from 8 mM to 3 mM. The presence of either urea or
ethanolamine was required, together with DTT, to control protein
aggregation."

Hope that this helps,

Michael

****************************************************************
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: garavito@msu.edu
****************************************************************

On Nov 13, 2007, at 4:09 PM, Joe wrote:
> The other possibility is 400 mM imidazole in the buffer. The
> precipitate looks like silk.
>
> On 11/13/07, Bryan W. Lepore wrote:
>> you didn't say how you know its protein - is it?
>>
>> interesting though.
>>
>

On Nov 13, 2007, at 4:13 PM, Joe wrote:

> The precipitate does not disappear automaticly if I don't add DTT.
> If it's the precipitate of imidazole, then why DTT can dissolve it?
> thanks
>
> On 11/13/07, Sanishvili, Ruslan wrote:
>> This may not apply in your case but it is not uncommon for a
>> protein to
>> "precipitate" in a microcrystalline shower when put in cold. Once it
>> worms up, the crystals dissolve and the precipitate clears up. It is
>> easy to check under a high magnification microscope.
>> Cheers,
>> N.
>>
>>
>> Ruslan Sanishvili (Nukri), Ph.D.
>>
>> GM/CA-CAT, Bld. 436, D007
>> Biosciences Division, ANL
>> 9700 S. Cass Ave.
>> Argonne, IL 60439
>>
>> Tel: (630)252-0665
>> Fax: (630)252-0667
>> rsanishvili@anl.gov
>>
>>
>>
>>
>> -----Original Message-----
>> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
>> Joe
>> Sent: Tuesday, November 13, 2007 2:30 PM
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [ccp4bb] DTT sensitive?
>>
>> Hi there,
>> I see enormous precipitate of my receptor protein when I take it
>> out of
>> freezer. But all the precipitate dissolved quickly after I added
>> 1mM DTT
>> to the solution. Does this mean that some surface Cys are causing
>> problem? Or why is the protein so sensitive to DTT?
>> Anybody experienced this kind?
>> Any advice is appreciated.
>> -Joe
>>
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: CCP4 Study Weekend cost increase from Sat. 17 Nov.
From: Charles Ballard c {- dot -} c {- dot -} ballard {- at -} DL {- dot -} AC {- dot -} UK
Date: 2007-11-14

Next message:
Subject: Fwd: Re: Protein powder diffraction (fwd)
From: Jonathan Wright wright {- at -} ESRF {- dot -} FR
Date: 2007-11-14