[ccp4bb] salt sensitive complex

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CCP4bb <-- 2008 <-- January 2008 <-- 22 January 2008

Previous message:
Subject: Re: differences between Rsym and Rmerge
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-01-22

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Subject: Why there are difference density when occupancy is 1.00
From: Sun Tang suntang2222 {- at -} YAHOO {- dot -} COM
Date: 2008-01-22

Subject: salt sensitive complex
From: Jerry McCully for-crystallizaiton {- at -} HOTMAIL {- dot -} COM
Date: 2008-01-22

Dear All:

Recently I am pursuing the crystallziation of a complex formd by two individual proteins and I met several interesting problems though they are kind of off-topic.

Any suggestions for these problems will be highly appreciated.

BIAcore showed about submicromolar affinity(both Kinetic and steady-state fitting) for these two proteins in the complex. However, precipitates immediately appeared when these two proteins were mixed together even at 10uM(<0.3mg/ml) concentration in the condition of low salt(less than 20mM NaCl).
By the way, these two proteins completely precipitated when the molar ratio is 1:1 in this condition.

THerefore, I increased the salt concentraion step by step and finally I can keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM NaCl(the minimum of salt concentration). Wierd thing happened when ITC experiments were carried out to confirm the binding affinity. 20uM in the sample cell and 200uM in the syringe could not give enough heat for a good curve fitting. The optimistic estimation of the affinity is lower than 5uM, which is much lower than the affinity given by BIAcore in the same buffer(25mM Tris plus 150mM NaCl).

Now I am suspecting the capability of the interaction between these two proteins. However, I can not explain why these two guys precipitated stoichiometrically if they do not interact with each other.

Is the complex salt-sensitive therefore there was just minor binding in the high-salt condition revealed by ITC?

I am planning to do the ITC again in the condition of 25mMTris and 60mM NaCl.

What if the affinity given by ITC is still much lower than that by BIAcore. Which one should I choose to believe?

Are there some better ways that I can validate the binding affinity?

Thanks again for your great ideas.

Jerry McCully

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CCP4bb navigation

CCP4bb <-- 2008 <-- January 2008 <-- 22 January 2008

Previous message:
Subject: Re: differences between Rsym and Rmerge
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-01-22

Next message:
Subject: Why there are difference density when occupancy is 1.00
From: Sun Tang suntang2222 {- at -} YAHOO {- dot -} COM
Date: 2008-01-22