Suppose I have, say, a zinc-structural metalloprotein. Can I ensure that the metal and protein will stay associated under not super gentle conditions (during electrophoresis, for example) by crosslinking the metal into the protein's tertiary structure?
Hi David,
I am dividing the answer into three distinct sections to enhance its clarity and helpfulness. Do let me know in case I missed something in the question that made me attempt the answer in this particular way:
1. In the technique of electrophoresis, usually the protein's tertiary structure is disrupted by boiling and reducing agents and it is linearized and prevented from refolding by using a detergent like SDS that coates the protein. Hence, to the best of my knowledge, I do not think that you can crosslink the metal onto the "tertiary structure" of protein during "electrophoresis" because there is no tertiary structure during the "not-so-gentle" procedure of electrophoresis.
2. Metal ions are usually bonded to protein by coordination bonds. Since crosslinking indicates the creation of covalent bonds, if my understanding is right, it is highly unlikely that metals can be crosslinked to proteins.
3. Unlike metal-dependent enzymes in which the metal is loosely associated with the protein and can easily fall-off, metalloenzymes are very tightly associated with their metal ions and copurify with them...
I hope the answer helps!!
Hi David,
I am dividing the answer into three distinct sections to enhance its clarity and helpfulness. Do let me know in case I missed something in the question that made me attempt the answer in this particular way:
1. In the technique of electrophoresis, usually the protein's tertiary structure is disrupted by boiling and reducing agents and it is linearized and prevented from refolding by using a detergent like SDS that coates the protein. Hence, to the best of my knowledge, I do not think that you can crosslink the metal onto the "tertiary structure" of protein during "electrophoresis" because there is no tertiary structure during the "not-so-gentle" procedure of electrophoresis.
2. Metal ions are usually bonded to protein by coordination bonds. Since crosslinking indicates the creation of covalent bonds, if my understanding is right, it is highly unlikely that metals can be crosslinked to proteins.
3. Unlike metal-dependent enzymes in which the metal is loosely associated with the protein and can easily fall-off, metalloenzymes are very tightly associated with their metal ions and copurify with them...
I hope the answer helps!!
If you performed a SDS PAGE as you denatured your protein using SDS, high temperature, it will not get the metal ion anymore for sure.
Unfortunately there is no crosslinker for metal ion and protein available yet.
David,
1) Some (rare) proteins can interact with their metals even under SDS-gel conditions. Thus, Calmodulin has drastically different mobility on the SDS-gel in the presence and absence of Ca2+.
2) You, however, should be interested in a more universal approach, such as native gel electrophoresis. There are numerous modifications of this technique (one of them is Native blue electrophoresis with coomassie brilliant blue playing a role of a mild, surface bound detergent). However, if your protein of interest is acidic (pKa < 7, better below 6.8), then you can use regular SDS-PAGE protocol except you i) should REMOVE SDS from ALL THE COMPONENTS (i.e. gels, running buffer, loading buffer) and ii) MAKE SURE TO NOT BOIL YOUR SAMPLES.
3) Metal cannot be "crosslinked" as if you succeeded in strengthening metal-protein coordination bonds by genetic manipulations you would be dealing with a very different metal-binding site; I would guess that binding of Metal can be enforced indirectly by stabilizing the native structure of protein e.g. by crosslinking protein elements around the metal binding site.
Best,
Dmitri
@ Most replies - I was thinking of native PAGE when I said gel electrophoresis, not SDS-PAGE, but thanks for the replies!
@ Dmitri - in response to 3) Yep, that's what I had in mind, I just wondered if it had been demonstrated
@Peter - Interesting, thanks!
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