Hi,
I am sorry that I cannot recall the
highest concentration of DTT that I ever used (inadvertently) when working with
disulphide-containing proteins. However I have the following mildly useful
suggestion:
If you have properly formed –S-S- as
well as a bunch of free –SH perhaps a safer strategy is to modify the
available –SH with something – it could be a reversible protective
group or a permanent one such as iodiacetamide. This way you avoid playing with
–S-S- shuffling entirely. Notably, this will reduce your chances of
obaining a derivative since protected –SR are much less likely to react
with heavy atom agents.
As a useful bonus, if you modify the SH
with e.g. iodoacetamide, then run MS on the intact protein – you will
find out how many –SH remain unmodified. Among those, some are in –S-S-
bonds and some are buried and therefore inaccessible to the reagent. By
repeating iodoacetamide treatment post-reduction or on an unfolded sample you
can further figure out the pattern.
Good luck,
Artem
“Nothing is built on stone; all is
built on sand, but we must build as if the sand were stone”
Jorge Luis Borges
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of Brad Bennett
Sent: Monday, August 10, 2009 5:06
PM
To: [log in to unmask]
Subject: [ccp4bb] Disulfide bond
"survival" in the presence of DTT?
Hi all-
Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide
bonds from forming between free Cys but to preserve native disulfides, I have
heard of using a very low (say < 0.5 mM) concentration of DTT. I've recently
come across a paper where the assignment of 3 disulfides in a protein structure
was carried out by calculating a 4A resolution anomalous difference map
(detecting the sulfur anomalous signal with 1.7A x-rays). However, at
every step in the protein prep and in the crystallization solution, there
exists mM concentrations of DTT. Every protein is different, so the
microenvironment and the protection of a disulfide will be different for every
one you come across. But I am surprised that a disulfide can
"withstand" that much DTT. I guess the proof is in the pudding, which
would be the anomalous peaks observed in the maps. What is the upper limit on
DTT (or B-ME or TCEP) concentration that people have used and still maintained
native disulfides?
Thanks-
Brad