If the mutation interferes with folding, yes, it may prevent the formation of correct disulfide bonds.
Dear Dr. Annemarie Honegger, thank you for your answer..can I prove that by any bioinformatic tool?I introduced a mutation in a disulfide bonded protein. now I see a double bond in SDS-PAGE gel. is it possible that my mutation affect the disulfide bond?
You can use CD, UV/Vis to investigate changes in the secondary structure of your protein.
You observe creation or destroy of the SS bond?
The simplest is probably circular dichroism. If you have access to MS facility, you can determine, whether Cys are involved in SS bond or not.
Dear Tomáš Hluska thank you alot.I didn't observe destruction of disulfide bond, I just saw a double bond on SDS-PAGE..I think that probably my mutation cause the destruction of disulfide bond in some proteins
Did you do a mutational addition of a cysteine that could act as a reducing agent and cause cleavage of a disulfide bridge, if that is what you are asking? Is the protein sample on the gel only the mutant protein and no other proteins? Did you run the sample reduced with reducing chamber buffer (NuPAGE gels)? Did you include an alkylating agent when running the sample non-reduced to block free thiols that cause mis-folding? If you did a cysteine deletion or a cysteine substitution then there may be an unpaired cysteine that acts inter molecularly causing disulfide exchange and therefore misfolding and aggregates. If the non-mutated protein has an uneven number of cysteines and you mutated the free cysteine then there could be dramatic refolding effects. A second band migrating close to another band may be a misfold or if it is migrating much higher than the other band then that may be an aggregate. If you run the sample reduced and get only one band then mis-folding may be the issue. Characterizing a putative misfold is another subject matter. It may also be possible that one of the bands is a clipped specie. Running the sample reduced versus non-reduced on the same gel in adjacent lanes is very simple, fast, and cheap to do in order to show the relationship between the two bands and if there's mid-folding.
Dear Dr. Grant Shimamoto thank you for your great accuracy and answer..my SDS-PAGE gel is attached..my mutation is not addition of any cystein..it is the substitution of Leu to Methionin.. but this mutation is in the vicinity of disulfide bond in the tertiary structure of the protein..i examined the SDS-PAGE of native protein in both reduced and non-reduced condition, but I didn't see a double bond similar to this case..I think this mutation may lead to misfolding of some proteins or destruction of disulfide bonds, but I didn't use any alkylating agent. but my SDS-PAGE is in reduced condition by 2-mercaptoethanol. and in non-reduced condition we see double bond, too.
Dear Zahra
Before seeing your gel:
I would not think that your result is due to a problem with S-S formation.
About bands in the gel: Is there the original protein and the mutant protein? If yo do not show both it is difficult to interpret the results. I just see two lanes showing the same whatever this would be.
dear Dr. Rafael Franco...both of two bands are mutant protein...original protein had a single bond on gel in both reduced and non- reduced condition
The story is becoming clearer. As you wrote: "examined the SDS-PAGE of native protein in both reduced and non-reduced condition, but I didn't see a double bond similar to this case." How many cysteines are there in this protein? Is the expression system mammalian (glycosyaltion) or E coli? If you have enough material for the biophysical characterization for both the wild type and mutant proteins then that may be the next step. If there's insufficient material then maybe gel work and mass spec will be fast and efficient as described next.
So the wild type protein doesn't show a doublet but just one band whether reduced or non-reduced, right? But is the reduced material band running slightly higher than the non-reduced material? Does the non-reduced wild type migrate to the same position as one of the two bands in the doublet of your non-reduced mutant protein? If it doesn't do this after knowing the mutant and wild type protein have essentially the same masses because of the conservative mutation and absence of any clipping, then there may be different ratios of SDS/protein and/or Stoke's radius differences for each band.
Are you absolutely sure the reducing agent is fresh and in high enough concentrations? TCEP may be a better reducing agent and iodoacetamide is a good alkylating agent to keep the cysteines blocked thereby prevent re-oxidation in situ (in the gel) which is why NuPage gels were made. You might want to run a positive control of a protein that demonstrates the gel shift reduced vs non-reduced, perhaps BSA. Just to be sure there is no significant mass difference before pursuing other explanations, is it possible to run MALDI mass spec or LC/MS since your sample looks clean enough for this? There should be only one parent +1 mass ion if both bands are the same in mass. If there's only one mass then would you have a strong case for arguing that the two bands differ on a gel because of conformational differences of in binding of SDS?
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