[ccp4bb] Selenomethionine reduction v.s. disulfide bond

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

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Subject: Re: Differentiating bound Mn & Ca.
From: Julie Bouckaert bouckaej {- at -} VUB {- dot -} AC {- dot -} BE
Date: 2007-04-16

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From: "Leslie A {- dot -} " andrew {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK
Date: 2007-04-16

Subject: Selenomethionine reduction v.s. disulfide bond
From: Tiancen Hu tony_htc {- at -} 126 {- dot -} COM
Date: 2007-04-16

Dear all,

Sorry for the non-CCP4 question.

We're working on selenomethionine substitution of a protein (4 Met/185 a.a.) containing two disulfide bonds. The protein was expressed in E.Coli. with non-reduced cytoplasm (OrigamiB). Now we're in a dilemma of deciding whether to add DTT during the purification of SeMet proteins. On one hand, we're informed to keep the SeMet as reduced as possbile during purification however risking damaging our disulfide bond. But on the other hand, some papers have reported that oxidized SeMet could enhance the anamolous signals. (Acta Cryst. D (2000) D56, 785-788, Oxidation of selenomethionine: some MADness in the method!; Acta Cryst. D (2001) D57, MAD on threonine synthase: the phasing power of oxidized selenomethionine). We've tested the effect of DTT on breaking down the disulfide bonds and found that even 50mM DTT can only reduce part of our protein at 37 degree celsius for 10 minutes. My question is that is oxidation destined to breakdown or cause instability of SeMet? Will it be f!
easible that we purify and crystallize the SeMet protein in a non-reduced enviroment just like our unlabelled native protein? Any suggestions, experience or references will be greatly appreciated! Thanks in advance!

Tiancen Hu
Shanghai Institue of Materia Medica
Rm. 2107, #555, ZuChongzhi Rd., PuDong
Shanghai 201203
P.R. China

CCP4bb navigation

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

Previous message:
Subject: Re: Differentiating bound Mn & Ca.
From: Julie Bouckaert bouckaej {- at -} VUB {- dot -} AC {- dot -} BE
Date: 2007-04-16

Next message:
Subject: imosflm bug for Mar 225 (3x3) detectors
From: "Leslie A {- dot -} " andrew {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK
Date: 2007-04-16