August 18, 2019, 05:18:20 AM
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Topic: Water soluble version of Cystine  (Read 2754 times)

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Offline Babcock_Hall

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Re: Water soluble version of Cystine
« Reply #15 on: December 17, 2018, 05:33:12 PM »
I am assuming that you are using cystine and not a peptide containing cystine.  The solubility of amino acids depends upon pH.  What pH is your experiment, and can it be changed?

Offline crystalguy

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Re: Water soluble version of Cystine
« Reply #16 on: December 17, 2018, 05:46:59 PM »
I am assuming that you are using cystine and not a peptide containing cystine.  The solubility of amino acids depends upon pH.  What pH is your experiment, and can it be changed?

Yes, Cystine only.  The pH needs to be around 7 to 8 for the protein to behave well.  I realize at very acidic conditions Cystine is more soluble, but that would kill the protein.

Need to add the Cystine at concentrations approaching 1 mM.

Offline Babcock_Hall

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Re: Water soluble version of Cystine
« Reply #17 on: December 17, 2018, 05:48:33 PM »
I would predict that cystine would be least soluble at its pI, which should be near 6.

Offline crystalguy

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Re: Water soluble version of Cystine
« Reply #18 on: December 17, 2018, 05:51:59 PM »
I would predict that cystine would be least soluble at its pI, which should be near 6.

Yes true, but even at pH 8 it's not very soluble unfortunately.

Offline hypervalent_iodine

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Re: Water soluble version of Cystine
« Reply #19 on: December 17, 2018, 06:21:54 PM »
Cysteine was an autocorrect typo on my part, apologies. I would still be very concerned that making any changes to your cystine molecule will change or abrogate binding in ways that you can’t predict (because you don’t know about how it binds). Especially if the changes you are making are for increasing solubility, since this would normally mean making the molecule more polar, and polarity changes like that could drastically alter binding. IMO, any results you might get from such a molecule would not really be that meaningful in terms of what you are trying to look at.

Do you know how much more cystine you can get into solution at 1% DMSO and how much excess you will have compared to enzyme concentration? It might be enough to get crystals. Have you been able to get crystals of the protein without cystine bound? I assume you probably have. Do you know if the structure changes when it binds to cystine, and could you possibly do any in silico modelling to get an idea of where it might bind?

Offline Babcock_Hall

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Re: Water soluble version of Cystine
« Reply #20 on: December 17, 2018, 09:24:33 PM »
https://pubs.acs.org/doi/pdf/10.1021/je9501853
"Solubilities of l-Cystine, l-Tyrosine, l-Leucine, and Glycine in Aqueous Solutions at Various pHs and NaCl Concentrations"

I imagine that you have seen this already.

Offline crystalguy

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Re: Water soluble version of Cystine
« Reply #21 on: December 30, 2018, 07:38:08 PM »
Cysteine was an autocorrect typo on my part, apologies. I would still be very concerned that making any changes to your cystine molecule will change or abrogate binding in ways that you can’t predict (because you don’t know about how it binds). Especially if the changes you are making are for increasing solubility, since this would normally mean making the molecule more polar, and polarity changes like that could drastically alter binding. IMO, any results you might get from such a molecule would not really be that meaningful in terms of what you are trying to look at.

Do you know how much more cystine you can get into solution at 1% DMSO and how much excess you will have compared to enzyme concentration? It might be enough to get crystals. Have you been able to get crystals of the protein without cystine bound? I assume you probably have. Do you know if the structure changes when it binds to cystine, and could you possibly do any in silico modelling to get an idea of where it might bind?

Yes you're right, the changes could be deleterious.  But it really is a trial and error process.  The main goal though is to get crystals of the protein, regardless of what the small molecule is doing which is minimally just stabilizing the protein/'tightening' it up. 

At 1% DMSO, we cannot get enough Cystine into solution because the affinity is quite weak, in the micromolar range.  We cannot get crystals of the protein alone and have no idea where it binds on the protein. 

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