Re: [ccp4bb] Cysteine oxidation product
Hey Dhiraj, I saw this email yesterday, and I was waiting for others to respond. This seems like there is no simple answer to your question. This would be advantageous, if you kindly share the chromatograms of the WT proteins, and the mutant one, both in the presence and absence of the reducing agent. Likewise, an image of Native PAGE, and the experiment conditions will help anyone to understand the complexity of the situation. Of course, you may hide the names of the proteins. Kind regards, -Z Zaigham M Khan, PhD Associate Scientist Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York United States On Sun, Nov 1, 2020 at 11:53 AM Srivastava, Dhiraj < dhiraj-srivast...@uiowa.edu> wrote: > Hi Jon >there is no surface exposed cysteine nearby which can > potentially make disulfide bond with the modified cysteine > intramolecularily. > > Dhiraj > -- > *From:* Jon Cooper > *Sent:* Sunday, November 1, 2020 7:41 AM > *To:* Srivastava, Dhiraj ; > CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] Cysteine oxidation product > > Hello, > > does the protein you added cysteine to (I assume you mean by mutagenesis) > have another cysteine which could make an intra molecular disulphide with > it? That would probably not show on non-reducing SDS? > > Best wishes, Jon Cooper > > > > Original Message > On 31 Oct 2020, 15:58, Srivastava, Dhiraj < dhiraj-srivast...@uiowa.edu> > wrote: > > > 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 > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Cysteine oxidation product
Hi Jon there is no surface exposed cysteine nearby which can potentially make disulfide bond with the modified cysteine intramolecularily. Dhiraj From: Jon Cooper Sent: Sunday, November 1, 2020 7:41 AM To: Srivastava, Dhiraj ; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cysteine oxidation product Hello, does the protein you added cysteine to (I assume you mean by mutagenesis) have another cysteine which could make an intra molecular disulphide with it? That would probably not show on non-reducing SDS? Best wishes, Jon Cooper Original Message On 31 Oct 2020, 15:58, Srivastava, Dhiraj < dhiraj-srivast...@uiowa.edu> wrote: 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Cysteine oxidation product
Hello, does the protein you added cysteine to (I assume you mean by mutagenesis) have another cysteine which could make an intra molecular disulphide with it? That would probably not show on non-reducing SDS? Best wishes, Jon Cooper Original Message On 31 Oct 2020, 15:58, Srivastava, Dhiraj wrote: > 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 > > --- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Cysteine oxidation product
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 Sent: Saturday, October 31, 2020 1:29 PM To: CCP4BB@JISCMAIL.AC.UK ; Srivastava, Dhiraj 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 on behalf of Srivastava, Dhiraj Sent: Saturday, October 31, 2020 4:58:37 PM To: CCP4BB@JISCMAIL.AC.UK 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] 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 on behalf of Srivastava, Dhiraj Sent: Saturday, October 31, 2020 4:58:37 PM To: CCP4BB@JISCMAIL.AC.UK 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
