Sulfhydryl reagents to protect S=S bond

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Tue Jun 26 17:55:18 EST 2001


Dear Wolfgang,
Thank you for your advise. DPDPB seems to be introducing too big an insert
between cysteines. My aim is to"conserve" a circular structure of the
peptide without altering its geometry too much.

NBD is also too big.

Obtaining peptide cyclized via S=S is no problem. We can order synthetic
peptides with any modifications from companies, and of course we can express
it in the periplasm so that the S=S will be preserved. The problem is that
when we use the peptide in our experiments with animal cells, S=S gets
degraded in the reduced intracellular environment as soon as the peptide
penetrated the cell.

Any ideas how to solve the problem?

Thanks.
Emir


----- Original Message -----
From: "Wolfgang Schechinger" <wolfsc at ibms.sinica.edu.tw>
To: "Emir Khatipov" <ekhatipo at midway.uchicago.edu>
Sent: Tuesday, June 26, 2001 2:11 PM
Subject: Re: Sulfhydryl reagents to protect S=S bond


: Emir,
:
: The solution probably depends on the nature of your protein. If
: there are no Args and Lys and -OH and you could live with a N
: terminal protected or modified derivative, many bidentate
: compounds that are reactive towards NH2 also are reactive
: towards SH and could do the job.
:
: Pierce has a lot of cross linkers in their catalogue. Check if
: DPDPB could work on your problem.
:
: Since NBD-F and NBD-Cl (Molecular Probes or Fluka) are
: suitable for SH labelling, a bifunctional F/Cl- derivative of both
: (e.g. F-NBD-CH2-NBD-Cl, there should be a way to have an
: aliphatic bridged derivative that itself also should be
: fluorescent), if not available, you might have it synthesized by
: your orgo chem colleagues as the ultima ratio if anything else
: fails.
:
: Or simply express the target protein on the surface of your
: cells (fuse it e.g. to the transmembrane domain of PDGFR,
: that's available as vector from some company, if you need it, i'll
: dig it out) and check for the binding.
:
: That's all,
:
: Wo
:
: Wolfgang







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