Though the samples were treated with TCEP and 1M Arg, protein aggregation still can be observed by NMR after 2-3 days.
To figure out why, I ran SDS-PAGE with different sample pretreatment:
protein+loading buffer（with SDS+β-Me) and protein+loading buffer(with SDS but no β-Me)..
The result is as shown below
Can it prove protein aggregation was induced by disulfide bond formation?(there are 2 cystein in the protein . C108 & C120)
Yes, it seems you have shown disulfide aggregation as assayed by SDS-PAGE, reduced versus non-reduced. It's good that you ran the reduced lane adjacent to the non-reduced lane because there's a diagnostic trick to do this that's important in further demonstrating your hypothesis. If you have sufficient protein loading and sufficient reducing agent in your sample then some of the reducing agent migrates laterally to partially reduce the proteins in the adjacent non-reduced lane. In your case, the resulting stained gel will show a streaking effect where, vertically, half of the non-reduced lane next to the reduced lane will have the dimer streaking down to the reduced monomer position and fusing with the side spur of the reduced monomer band. This demonstrates the relationship between the two bands and is very helpful in refolding studies. You might increase the b-ME concentration of your sample buffer or make fresh reducing sample buffer.
The sharpness of the reduced band is consistent with the reduced and denatured conformation usually see with reduced proteins on SDS-PAGE. Seeing similar morphology and gel position of the monomer band in the non-reduced lane seems to indicate that your bulk lot of protein is still reduced and of course with a small percentage of dimer. If the monomer were oxidized then the migration might be slightly lower than the reduced monomer in context of the 12kD mass (gel shift assay) due to the change in Stokes radius. By the way, the TCEP should still be very effective after this length of time and your results seem to indicate this. If your protein is soluble at acidic pH's then you can protonate the sulfhydryl functions and greatly slow down the oxidation in terms of formulation and storage. TCEP is also still extremely effective at acidic pH's.
MALDI may be a quick mass spec method to confirm the relationship between the two bands in question if your sample is really this pure. You should see the +1 parent ion of the dimer and a small amount of its +2 m/z vedry near or superimposed upon the +1 parent ion of the monomer mass. The sinapinic adduct of the monomer however may mask detection of the +2 ion of the dimer. Triple quad LC/MS will be definitive but take a little more time and development.
What is your purpose on this? crystallization?
I assume that you ran the soluble fractions of your samples. Theoretically, this gel does prove aggregation due to S-S bond formation at a very mild extend.
But it would not tell you if your protein is present as monomer/dimer and i would suggest running a native PAGE gel (same as SDS-PAGE but without SDS at every single steps).
Did you want to go beyond the disulfide question and know what the disposition of your protein is in your current storage buffer? You would then need an orthogonal method of which there are many: SEC, pull down, analytical ultracentrifugation, your NMR, DLS, etc.
Thank you, Grant Shimamoto
The monomer should be 13465 Da, theoretically. And, it seems nearly 30kDa when I run gelfiltration. So I speculated it should be a dimer.
I have been puzzld why it seems so small in SDS-PAGE until see your description about Stokes radius.
About pH, pI of protein is 5.6, and protein would be precipated when pH＜6. So I can't make buffer acidic and the buffer for protein is 20mM HEPES,100mM NaCl,1M Arg,1mM TCEP (pH7.0).
And about the MALDI, I will talk about it with my mentor later.
For my purpose, I want to use NMR to get the structure. However, NMR has no advantage for protein more than ~40kDa. So I hope the protein could be maintained as a dimer. And, do you think it's feasible if I increase the con. of TCEP.
Besides, I forget it before. My protein is a Metalloprotein with Cu or Fe. I don't know if the meal ion will weaken the effect of TCEP.
Thank you for your suggestion， sincerely.
Hi there,
Obviously there is disulfide bond formation between monomers to form a dimer. Why do you suggest the aggregation you see by NMR is promoted by interchain disulfide bond formation? If aggregation occurs in presence of TCEP it rather suggests disulfide bond formation would not be involved in the process... There are several issues to look at before concluding: what is the actual oligomerization state of the protein in solution in presence and in absence of reducing agent(SEC will tell you)? What happens if cysteines are mutated into serine? Does it prevent dimerization and/or aggregation? What is the effect of metal on aggregation, dimerization aso? And finally what about Arginine concentration? 1M is a lot and tends to maintain soluble proteins prone to aggregation...
Do you want the dimer or the monomer?
Metal ions such as nickel do not affect TCEP.
I've rarely seen proteins precipitate at their pI. I believe this was an observation seen long ago for a few proteins but then became a general rule rather being an exception. However, proteins are less soluble at their pI but that doesn't mean they will precipitate. I've asked many people to adjust their samples to a pH equal to the pI of the protein and they are quite surprised to see that their protein didn't precipitate. Try pH 4, 4.5, 5, 5.5, and 6.7and see what happens. If it's OK then you might be able to use less TCEP but more TCEP would be OK
Do the cysteines complex with the metal ions? I assume the metal ions are already present?
Or do the cysteines form an intra-protein disulfide or does one or both cysteines form an inter disulfide bond to create a covalent homodimer.? In other words, are the cysteines supposed to be oxidized to generate a correctly folded molecule?
If essentially all the protein material is dimeric by SEC but monomeric by non-reducing SDS-PAGE then are you are you claiming that the protein is supposed to be a non-covalent dimer but has a slight contamination of covalent dimer due to insufficient concentration of TCEP for this length of time and pH? What happens when the cysteines are alkylated with iodoacetamide or n-ethylmaleimide?
Hi Bin Liu,
maybe silly question but I've got to ask, do you know if the monomeric form of your protein have an S-S bond? Have you built a homology model of your protein?
My obvious guess is that there is a S-S bond in the monomeric form and the dimeric state is non-covalently bound of the 2. That would explain the very small amount of dimer (as the extra band) in the SDS-PAGE pic i.e. or else it would be much thicker "extra" bands.
How well is your protein's stability after 1 - 7 days of storage? I have not done NMR myself so I couldnt help much, but can the solution for NMR containing the beta-ME?
Hi Grant Shimamoto
The protein is a metallprotein and it’s blue during purification, so the metal ion is already there. Due to its superfamily, it should be HxxxHxxE which complex with metal.
About pH, I have already tried pH 3,4,5,6, and it would be precipated. that's why I choose pH=7.
I can't get monomeric, dimer is ok for study but can't be more. I predicted there is hydrophobic polymeric sites LYLLV by PASTA2.0(http://protein.bio.unipd.it/pasta2/). I have tried to mutate some amino acid, but they all made protein insoluble.
I‘m not 100% sure there is no intra-disulfide, I just did some prediction and all the analysis points to reduced. Moreover, I can only see the monomeric band if I run a PAGE after SEC immediately. I have mutated cysteine, and let's see what will happen.
Furthermore, I will try increase the con. of TCEP or alkylate with sth if it doesn't affect its structure and activity.
Hi Dominique Liger
I'm not sure the aggregation observed by NMR was induced by disulfide. That's why I ran this reduced and non-reduced PAGE. The con. of Arg is good. Without Arg, the protein completely changed in Native-PAGE in 2~3 days.
By SEC, the protein is a dimer in presence or absence of reducing agent.
I have mutated cysteine to serine, but it make protein insoluble now.
Hi Binh (Albert) An Nguyen
I have build a homology model of my protein and cysteines are reduce. The extra band in SDS-PAGE get thicker with time and I want to increase the con. of TCEP next time for someone tell me metal ion may be weaken the effect of reducing agent.
The stablity of protein is not perfect but acceptable.
No problem for B-Me in soultion for NMR, but it's not suitable for long-term NMR data collection.
Your results seem to be consistent with the gel shift commentary made earlier (monomer position of reduced vs non-reduced being the same). So your protein seems to be in the fully reduced state except for a trace of oxidized dimer which may be mis-folded. If you dialyze out the TCEP quickly and then test the resulting protein solution with DTNB then there should be a positive reaction for reduced proteinyl cysteines.
Also, the earlier supposition that your protein is a non-covalent dimer by SEC and becomes monomeric by disruption through SDS in the gel seems to also be consistent but you think there is no coordination of the copper by the thiols. Is it possible that that the copper forms a a Biuret complex with the peptide bonds rather than or in addition to HxxxExx complexation because Biuret is also blue at alkaline pH; is there a wavelength shift? Can Biuret be a dimeric complex involving two protein molecules?
The earlier suggestion about nickel not reacting with TCEP and being barely sufficient to keep the protein reduced at the pH and time length you use was a reason to increase the TCEP concentration, maybe to 4 mM. The earlier suggestion also proposed that ferric, ferrous, cupric, and cuprous ions would unlikely react/coordinate with TCEP because of not having the electrons to donate to the d and f orbitals. TCEP utilizes water molecules. The iron may also already be reduced in the case of Biuret formation.
A question does remain. Are you absolutely sure that Cys 108 and 120 are not supposed to be oxidized to a disulfide? The monomer may still be mis-folded especially when it is not soluble at the acidic pH's and irregardless of the protein's pI. Needing arginine and TCEP for solubility/stability for a mammalian protein is also not a good sign for proper folding. Mis-folding may be an explanation for difficulty in crystallization and many other operations. If the protein has a putative cell-based functional assay or a binding assay and you feel it is correctly folded then you could test it functionally? Then again this may be a novel protein for which there is no native protein or standard available.
Do the following steps:
1)The free SH measured by introducing DTNB now
2)Then with the help, TCPE with guanidinium hydrochloride or DTT do the test again DTNB
3) Gel tricine-tris, native
4) Qualitative and Quantitative test results to show aggregation with disulfide bonds.
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