a. As protein folding generally starts from N terminus. If hypothetically I delete some parts of its N-terminus, will it still be stable and able to fold properly?
b. If I delete 30 amino acids from N-terminus the protein is not stable, but if I delete 60 amino acids (including the first 30) from N-terminus the protein becomes stable. What might be the reason behind?
probably the first 60 amino acids makes a full domain. So if you cut in the middle of this domain then the protein can not fold properly and thus becomes unstable.
Hi Tias, check out the following review manuscript about the N-end Rule & protein stability.
Article The N-end rule pathway and regulation by proteolysis
First, you need to describe your definition of your protein "stability" in your understanding. Is it the protein enzymatic activity, methabolic activity, immunologic activity, just mass-spec, or resistance to proteolytic degradation, you need just to specify that. Each protein is unique, some of them can still maintain their functions after truncation of their first 30 aa from their N-terminus (or loose the function), some other proteins can still have their functions after deleting their 60 aa from the N-terminus. If you are interested in immunological picture of these manipulations that would be more complicated and unpredictable.
Hello Tias,
Do you mean degradation when you over-express the protein or when you do the proteolytic digestion in vitro? By the way the loss of function or stability is not unique to N terminus. I had a experience working with DnaB protein of helicobacter pylori. So if I delete 29 amino acids from C terminus then the enzymatic activity of the protein was completely gone even though the motifs for enzymatic activity are far away . Moreover the solubility of the protein was also severely affected and it CD spectra it look like aggregation, please see my publication http://nar.oxfordjournals.org/content/35/9/2861.short).
Dear Ram Gopal,
Thanks for your suggestion. In my case I just want to over express the protein and check whether my constructs are fine or not. But the uncanny thing is a higher deletion mutant, A (deletion of 60 amino acids from N-terminus) is expressing but smaller deletion mutant, B (30 amino acids from N-terminus) isn't. I'm unable to understand that those 30 amino acids are deleted in A as well still its expressing. So what could be the problem with B's expression?
Protein folding involves the formation of disulfide bonds by partner Cys residues. Deletion of 60 amino acids in mutant A might have removed a pair of Cys residues but the rest of the protein can still fold properly and remain stable. On the other hand, deletion of 30 amino acids in mutant B might have removed one Cys residue, leaving its partner Cys ‘hanging’ that might lead to the formation of intermolecular disulfide bridge. Also, because of the possible removal of an intramolecular disulfide bond at the N-terminal region in mutant B, the remaining amino acids in this region might be more susceptible to proteolytic degradation.
Hi Tias, The review reference was: The N-end rule pathway and regulation by proteolysis, Alex Varshavsky, Protein Science 2011 20:1298.
But with further thought, I realize that when you are deleting the N-terminus, you are presumably maintaining the initiating Met residue. N-end Rule doesn't apply without cleavage.
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