pI under denaturing conditions - Plzz reply quick..Tkss! (Jan/18/2006 )
Hi all!
I'm looking for a standard mixture for IEF under denaturing conditions, e.g. 8M urea. Many companies sell protein standards for isoelectric focusing (IEF), but it seems that nearly all of the products are intended for NON-DENATURING IEF of native proteins.
My question is, can any one tell me where I might buy a standard mixture for denaturing IEF, or perhaps recommend a selection of known proteins which could provide nice bands in 8 M urea ?
Also, may be a stupid question.. (but since i m seeing a different pattern of the marker for native IEF under denaturing conditions.. i gotta ask it).. WILL THE pI of a protein be different in native and denaturing conditions??
Plzz do reply:)
Thanks!
-cheeztoast-
Sigma apparently sells a marker kit specifically designed for denaturing conditions (~3/4 down the page, "2D Electrophoresis markers"). I have no practical experience with it, though.
In theory, pI should not change dramatically with folding state. Unless you're dealing with membrane proteins specifically, those residues which have the greatest impact upon that property will invariably be on the outside of the molecule in the folded state, anyway, so exposing the inside should make no difference.
That isn't to say there can't or won't be a change in any given protein species, and other factors may be involved - if your standard marker looks different, there's clearly something else going on. It could be an interaction with your gradient or differing solution density that affect migration patterns. If you want to know for sure, I'd run side-by-side gels of 8M Urea vs 6M Guanidine. If you see the same effect, its probably a denaturing effect. If they look different, there may be something else involved.
-aludlam-
QUOTE (aludlam @ Jan 19 2006, 01:42 AM)
Sigma apparently sells a marker kit specifically designed for denaturing conditions (~3/4 down the page, "2D Electrophoresis markers"). I have no practical experience with it, though.
In theory, pI should not change dramatically with folding state. Unless you're dealing with membrane proteins specifically, those residues which have the greatest impact upon that property will invariably be on the outside of the molecule in the folded state, anyway, so exposing the inside should make no difference.
That isn't to say there can't or won't be a change in any given protein species, and other factors may be involved - if your standard marker looks different, there's clearly something else going on. It could be an interaction with your gradient or differing solution density that affect migration patterns. If you want to know for sure, I'd run side-by-side gels of 8M Urea vs 6M Guanidine. If you see the same effect, its probably a denaturing effect. If they look different, there may be something else involved.
In theory, pI should not change dramatically with folding state. Unless you're dealing with membrane proteins specifically, those residues which have the greatest impact upon that property will invariably be on the outside of the molecule in the folded state, anyway, so exposing the inside should make no difference.
That isn't to say there can't or won't be a change in any given protein species, and other factors may be involved - if your standard marker looks different, there's clearly something else going on. It could be an interaction with your gradient or differing solution density that affect migration patterns. If you want to know for sure, I'd run side-by-side gels of 8M Urea vs 6M Guanidine. If you see the same effect, its probably a denaturing effect. If they look different, there may be something else involved.
Thanks a ton for that! I've ordered for it.. lets c.. will let ya know of course:)
Besides, about pI - what about the hidden charges due to ionic kinda interactions.. all of that wudnt be possible under unfolded state of the the protein, right? (Dramatically, u said? But it would change right.. I m getting confused with the definition of pI.. they say net charge of protein ZERO.. or apparent net charge.. depending on how it is folded?)
Also i've come up with something else.. protein folding would be affected with pH too.. so not majorly but we do end up altering the state of protein under a pH gradient that we cant avoid using under native conditions too. Besides, at pI the protein may not be in its native state (saying that coz i know most proteins would clump and precipitate at their pI).. So how much would a change in pH affect protein folding? (Do u have something to read on that?)
Now, i m trying to load known proteins.. (calculating their pI with a software that would consider all charges) and see my protein's relative position.
Next probably i cud see with GuHCl but i dont think gels with GuHCl run fine.. smearish neh!?
Thanks again:)
Do write..
-cheeztoast-
QUOTE
Besides, about pI - what about the hidden charges due to ionic kinda interactions.. all of that wudnt be possible under unfolded state of the the protein, right? (Dramatically, u said? But it would change right.. I m getting confused with the definition of pI.. they say net charge of protein ZERO.. or apparent net charge.. depending on how it is folded?)
Studies of the dynamics of protein folding (solution proteins, NOT membrane proteins) have shown that protein folding is largely driven by hydrophobic interactions - the desire of phobic residues to be out of a polar environment far outweighs the desire of polar and charged residues to fulfill valence shells and equalize charge (they can largely do so by making contact with water and/or salts). Hence, the interior of a protein is going to be almost entirely hydrophobic. Those few residues that aren't are most likely going to be "paired up" with an oppositely-charged residue on the inside, so the effect of exposing them to solvent will be a net change of nearly zero.The "dramatically" caveat is that such interior pairings are dependent on the local environment, and pKa's are not going to oppose each other equally when both become exposed. So you may see a slight shift in pI by unfolding a protein. But in practice, adding and averaging all such pairings/groupings in a single protein will usually leave you with no change.
As with all things scientific, though, I'm sure there are exceptions.
QUOTE
Also i've come up with something else.. protein folding would be affected with pH too.. so not majorly but we do end up altering the state of protein under a pH gradient that we cant avoid using under native conditions too. Besides, at pI the protein may not be in its native state (saying that coz i know most proteins would clump and precipitate at their pI).. So how much would a change in pH affect protein folding? (Do u have something to read on that?)
It's true, proteins tend to be at their least soluble when the pH matches their pI. That doesn't necessarily mean they have changed their folding, it just means there are fewer surface charges available to stabilize it in solvent. In some cases that could lead to localized unfolding, but in most cases it just means the proteins aggregate together to escape solvent and stabilize their surfaces. Any structural changes that take place are likely limited to surface loops or mobile domains - melting and structural rearrangement will probably not happen because, once again, the stability of a fold is largely determined by its interior, hydrophobic environment, not surface charge.
Of course, if you're talking about extreme (ie, nonbiological) environments, then the model starts to break down - at low pH, you protonate nearly everything, and the thermodynamic stability of the hydrophobic interior is negated by the desire of different parts of the molecule to get away from each other. Not sure when that would start to take effect, nor whether it would matter in the particular experiment you're interested in - IEF is an equilibrium method, so whether or not it becomes unfolded shouldn't matter, so long as the unfolded species is still capable of passing through the gel matrix.
-aludlam-
The value for pI that i wud get if i used a software based on amino acid sequence to calculate protein pI.. say, expasy.. it would be under denaturing conditions, right?.. with most amino acids exposed.
Havent been able to conclude yet.. waiting for the marker.
Ran BSA and lysozyme with the expasy calculation.. but how do i know they matched the theoritical value?
QUOTE (aludlam @ Jan 20 2006, 01:25 AM)
QUOTE
Besides, about pI - what about the hidden charges due to ionic kinda interactions.. all of that wudnt be possible under unfolded state of the the protein, right? (Dramatically, u said? But it would change right.. I m getting confused with the definition of pI.. they say net charge of protein ZERO.. or apparent net charge.. depending on how it is folded?)
Studies of the dynamics of protein folding (solution proteins, NOT membrane proteins) have shown that protein folding is largely driven by hydrophobic interactions - the desire of phobic residues to be out of a polar environment far outweighs the desire of polar and charged residues to fulfill valence shells and equalize charge (they can largely do so by making contact with water and/or salts). Hence, the interior of a protein is going to be almost entirely hydrophobic. Those few residues that aren't are most likely going to be "paired up" with an oppositely-charged residue on the inside, so the effect of exposing them to solvent will be a net change of nearly zero.
The "dramatically" caveat is that such interior pairings are dependent on the local environment, and pKa's are not going to oppose each other equally when both become exposed. So you may see a slight shift in pI by unfolding a protein. But in practice, adding and averaging all such pairings/groupings in a single protein will usually leave you with no change.
As with all things scientific, though, I'm sure there are exceptions.
QUOTE
Also i've come up with something else.. protein folding would be affected with pH too.. so not majorly but we do end up altering the state of protein under a pH gradient that we cant avoid using under native conditions too. Besides, at pI the protein may not be in its native state (saying that coz i know most proteins would clump and precipitate at their pI).. So how much would a change in pH affect protein folding? (Do u have something to read on that?)
It's true, proteins tend to be at their least soluble when the pH matches their pI. That doesn't necessarily mean they have changed their folding, it just means there are fewer surface charges available to stabilize it in solvent. In some cases that could lead to localized unfolding, but in most cases it just means the proteins aggregate together to escape solvent and stabilize their surfaces. Any structural changes that take place are likely limited to surface loops or mobile domains - melting and structural rearrangement will probably not happen because, once again, the stability of a fold is largely determined by its interior, hydrophobic environment, not surface charge.Of course, if you're talking about extreme (ie, nonbiological) environments, then the model starts to break down - at low pH, you protonate nearly everything, and the thermodynamic stability of the hydrophobic interior is negated by the desire of different parts of the molecule to get away from each other. Not sure when that would start to take effect, nor whether it would matter in the particular experiment you're interested in - IEF is an equilibrium method, so whether or not it becomes unfolded shouldn't matter, so long as the unfolded species is still capable of passing through the gel matrix.
-cheeztoast-