Hi Matthias
I haven’t measured the affinity in a quantitative way. My
assumption of affinity increase is based on gel filtration. With reducing
agents, i don’t see comigration of two proteins on gel filtration
chromatography while without reducing agents, two proteins are co-migrating.
Dhiraj
________________________________
From: Barone, Matthias <[email protected]>
Sent: Saturday, October 31, 2020 1:29 PM
To: [email protected] <[email protected]>; Srivastava, Dhiraj
<[email protected]>
Subject: Re: Cysteine oxidation product
Hi Dhiraj
how did you measure the affinities? What are the two affinities, incl standard
error (or even amount of data points you used in the titration exp)?
Im just asking because Im curious as what you mean by "increase". If you add
DDT to an ITC for example, this will create a lot of background signal which
has to be taken into account. Did you make background titrations for both?
Dr. Matthias Barone
AG Kuehne, Rational Drug Design
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284
________________________________
From: CCP4 bulletin board <[email protected]> on behalf of Srivastava,
Dhiraj <[email protected]>
Sent: Saturday, October 31, 2020 4:58:37 PM
To: [email protected]
Subject: [ccp4bb] Cysteine oxidation product
Hi All
I added an extra cysteine residue to one of my protein interactions
partners and somehow it increased the affinity in the absence of reducing agent
however in the presence of reducing agent, the affinity is same. there is no
disulfide bond formation between the two interaction partner as in non-reducing
SDS-PAGE, I see the molecular weights corresponding to only the individual
proteins and not the sum of two.
Does anyone has ever seen this? at pH 8.0, what is the probability that a
surface exposed cysteine will have a net negative charge and still not very
reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there any
such example in literature that anyone knows?
Thank you
Dhiraj
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