Just a quick self-correction, the P(+3) in the previous post should be P(-3) -- I type too fast :) and of course, while the final product of this reaction is P(+5) the likely salient intermediate product is in fact P(-1) in the form of R3P=O (which undergoes further interesting reactions.
Artem On Sun, Apr 17, 2011 at 2:14 PM, Nian Huang <[email protected]> wrote: > Thank you for such great explanation. Hopefully people won't consider me > hijacking this thread. > Nian > > On Sun, Apr 17, 2011 at 11:08 AM, Artem Evdokimov < > [email protected]> wrote: > >> TCEP relies on a completely different chemistry to achieve the same goal >> as BME or DTT. >> DTT/BME use S(-)>SS with redox potentials of -0.26 to -0.33 V (at pH 7) >> whereasTCEP uses P(3+)->P(5+) oxidation with redox potential that's a lot >> higher (I don't know of a reference with a stated redox potential for this >> system) because TCEP readily reduces oxidized forms of both DTT and BME in >> solution. TCEP also does not readly break S-Hg bonds unlike DTT or BME. >> >> TCEP does not readily react with oxygen in solution (both DTT and BME >> react rapidly) and has long shelf life as buffered 1M solution at pH >> 5.6-6.3. >> >> TCEP has two non-trivial disadvantages that are for the most part not >> relevant for the purposes of crystallography: one is that it does not work >> very well in high Phosphate concentration, and the other is that it hinders >> the reaction of thiols with haloacetamides and suchlike (because it itself >> can react with haloacetamides). If you're labeling with haloacetamides you >> may want to use tri- tertbutul phosphine instead (it's much less soluble >> than TCEP, but 1 mM solution in water can be made). >> TCEP does not permeate biological membranes and therefore has been used to >> reduce thiols outside the cell while keeping intracellular ones intact. Due >> to its size and charge, it also is quite selective which protein disulphides >> are readily reduced - ones on or near protein surface are reduced quickly >> whereas buried or shielded ones are often not reduced at all w/o the use of >> a chaotrope. That's rather useful to us as it often allows us to reduce the >> unwanted inter-molecular disulphides (bad: aggregates) while at the same >> time preserve the valuable intra-molecular ones (good: structure) >> >> Artem >> On Sat, Apr 16, 2011 at 11:46 PM, Nian Huang <[email protected]>wrote: >> >>> Dear Horacio, >>> How does TECEP compare to BME or DTT? People claim it is better, but I >>> want some crystallographers' opinion? >>> >>> Nian >>> >>> On Sat, Apr 16, 2011 at 4:24 PM, Horacio Botti <[email protected]>wrote: >>> >>>> Dear Mike >>>> >>>> BME readily autooxidizes (need for metal traces and dissolved O2). Is >>>> yours a metalloprotein? Is your buffer contaminated with metals? Those >>>> situations would make the case a bit different. If not, unless your BME >>>> stock is already oxidized, blocking of the accesible thiols with BME should >>>> take some time. If you treat your protein for 40 min with fresh BME you >>>> should not observe thiol blocking. If you let the preparation to stay for >>>> several days, even at 4-6 °C you may observe the blocking that you may be >>>> observing. >>>> >>>> If you want to prevent Cys blocking you can also change to DTT (it is a >>>> dithiol, does not readily form mixed disulfides) and use it with caution >>>> (for thiol reduction it is advisable to use stoichiometric DTT (with >>>> respect >>>> to the number of Cys you need to reduce) and 10 fold excess of BME, look >>>> for >>>> their redox potentials). Take care of not "over-reducing" your protein if >>>> internal disulfide bonds are expected. Once reduced I suggest you to remove >>>> any reducing agent and store the protein at -80 °C. >>>> >>>> External Cys can be easily oxidized, they are highly expossed to metals >>>> and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster >>>> than SS bond formation, although some cys react at almost >>>> diffusion-controlled rates with oxidants (is yours a thiol'dependen t >>>> peroxidase?) You can take a look at the following reference (advertising): >>>> >>>> 2011. Factors Affecting Protein Thiol Reactivity and Specificity in >>>> Peroxide Reduction. Chem Res Toxicol. >>>> >>>> Metals can contaminate bad quality materials (water, salts, buffers, >>>> etc), take care of that too. If you need to control the redox state of your >>>> protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure >>>> accesible reduced thiol groups. >>>> >>>> Good luck! >>>> >>>> Horacio >>>> >>>> >>>> >>>> >>>> Quoting Kendall Nettles <[email protected]>: >>>> >>>> We see BME adducts in all of our estrogen receptor structures, though >>>>> we don't always put them in the models. Sometimes we only see one or two >>>>> atoms of the adduct, and in others it is completely ordered. We only see >>>>> it >>>>> on the solvent accessible cysteines. We do it on purpose. We used to >>>>> treat >>>>> the protein with iodoacetic acid to generate uniform modification of the >>>>> cysteines, but then we realized we could get then same homogeneity with >>>>> 20-50mM BME. >>>>> >>>>> Kendall Nettles >>>>> >>>>> On Apr 15, 2011, at 4:09 PM, "Michael Thompson" <[email protected]> >>>>> wrote: >>>>> >>>>> Hi All, >>>>>> >>>>>> I was wondering if anyone knew whether or not it is possible for >>>>>> reducing agents with thiol groups, such as DTT or beta-mercaptoethanol >>>>>> (BME), to form covalent S-S bonds with Cys residues, particularly >>>>>> solvent-exposed Cys? I have some puzzling biochemical results, and in >>>>>> the >>>>>> absence of a structure (thus far), I was wondering if this might be >>>>>> something to try to control for. I have never heard of this happening >>>>>> (or >>>>>> seen a structure where there was density for this type of adduct), but I >>>>>> can't really think of a good reason for why this wouldn't happen. >>>>>> Especially for something like BME, where the molecule is very much like >>>>>> the >>>>>> Cys sidechain and seems to me like it should have similar reactivity. >>>>>> The >>>>>> only thing I can think of is if there is a kinetic effect taking place. >>>>>> Perhaps the rate of diffusion of these small molecules is much faster >>>>>> that >>>>>> the formation of the S-S bond? >>>>>> >>>>>> Does anyone know whether or not this is possible, and why it does or >>>>>> does not happen? >>>>>> >>>>>> Thanks, >>>>>> >>>>>> Mike >>>>>> >>>>>> >>>>>> >>>>>> >>>>>> -- >>>>>> Michael C. Thompson >>>>>> >>>>>> Graduate Student >>>>>> >>>>>> Biochemistry & Molecular Biology Division >>>>>> >>>>>> Department of Chemistry & Biochemistry >>>>>> >>>>>> University of California, Los Angeles >>>>>> >>>>>> [email protected] >>>>>> >>>>> >>>>> >>> >> >
