Currently I am working on a reductase protein which I suspect may contain a FAD cofactor. However, I dont detect FAD in this protein after purification, then can I say this protein does not need a FAD to function properly?
I doubt this because I expressed this MOUSE protein in E. coli, even though many flavoproteins still have FAD after purification when they are expressed in E. coli.
I think I'd better also try to incorporate FAD into the protein and check whether it exhibits better activity.
No, by itself, the lack of FAD in the purified reductase does not demonstrate that your enzyme is not a flavin-dependent reductase. It is not at all rare for flavoenzymes overexpressed in E. coli to purify as the apo-protein. This sometimes has something to do with expression being too rapid to properly synthesize the holo-enzyme. Sometimes people try adding flavin to the bacterial medium, but that rarely (if ever) helps - E. coli can express very high levels of flavoproteins, so flavin biosynthesis has the capacity to keep-up. Try expressing your reductase at lower temperatures, and maybe use a different E. coli strain, like Rosetta.
Sometimes flavins are held only loosely and dissociate during purification; sometimes engineered affinity tags cause the problem. It occasionally helps to include free FAD in the purification buffers. Some apo-proteins are stable enough to be purified and will bind added flavin after the fact, while others don't. You're right to try adding FAD. Incubate your apo-enzyme with FAD on ice for at least a half-hour. Then remove the free FAD by gel-filtration. If you're lucky, you should get a nice yellow high-molecular weight fraction.
You didn't mention what your reductase is or why you expect it to be a flavoenzyme. If it really is a flavoenzyme, there might be a tiny amount of FAD - too little to see - bound to your preparations. Thus your sample may have low residual activity detectable by your assay, but the specific activity would be very low.
CD is not likely to be helpful here; it could be useful for other applications. CD is much less sensitive than absorbance spectroscopy, so it's only really useful when most of the enzyme has flavin bound, i.e., after you solve your immediate problem. We've collected CD spectra from 300 nm - 700 nm of a few dozen oxidized flavoproteins and they vary wildly; sometimes the signals are strong, sometimes not.
As for a "U-shaped conformation" of FAD... That's an appropriate description for a few flavoenzyme structures, such as DNA photolyase. And FAD folds over on itself in aqueous solution due to the hydrophobic interactions between the isoalloxazine and adenine ring systems. In most cases, however, FAD bound to proteins adopts a relatively extended conformation, with the isoalloxazine and adenine ring systems being quite distant.
Use Circular Dichroism in the Investigation of Protein Structure and Function
FAD being non-protein chromophores can give rise
to CD signals which depend on the precise environment of the chromophore concerned
The oxidised form of the free flavins shows two
absorption peaks at 360 nm and 450 nm, whereas
the reduced form shows a broad absorption over
the range from 300 nm to 500 nm. The neutral
semiquinone shows a characteristic weak
absorption around 600 nm, whereas the anionic
semiquinone shows strong absorbance at 380 nm
If your specific activity goes up after adding FAD, then I would say it is required for activity.
FAD in most cases is tightly bound to the enzyme/protein. Exceptionally, some can work with lose connection with protein. But, if it is essential for the functioning of the protein, the biological function of that protein will be lost. So, study of bioactivity/enzyme activity in absence and in presence of FAD will help you understand whether it needs FAD or not. If needed, then you can go for CD studies for the nature of binding.
Frankly, Dr.Md Rezaul Karim, I dont agree with you. In your comment you said " Exceptionally, some can work with lose connection with protein." I dont understand what you mean by term "loose connection?. What ever I know about FAD is FAD binding to the enzyme show essentially a single U-shaped conformation of this cofactor which, so far, is unique among FAD-carrying proteins. There is no tightly bound or loosely bound FAD. Dr.Md Rezaul Karim pls correct me if I am wrong.
No, by itself, the lack of FAD in the purified reductase does not demonstrate that your enzyme is not a flavin-dependent reductase. It is not at all rare for flavoenzymes overexpressed in E. coli to purify as the apo-protein. This sometimes has something to do with expression being too rapid to properly synthesize the holo-enzyme. Sometimes people try adding flavin to the bacterial medium, but that rarely (if ever) helps - E. coli can express very high levels of flavoproteins, so flavin biosynthesis has the capacity to keep-up. Try expressing your reductase at lower temperatures, and maybe use a different E. coli strain, like Rosetta.
Sometimes flavins are held only loosely and dissociate during purification; sometimes engineered affinity tags cause the problem. It occasionally helps to include free FAD in the purification buffers. Some apo-proteins are stable enough to be purified and will bind added flavin after the fact, while others don't. You're right to try adding FAD. Incubate your apo-enzyme with FAD on ice for at least a half-hour. Then remove the free FAD by gel-filtration. If you're lucky, you should get a nice yellow high-molecular weight fraction.
You didn't mention what your reductase is or why you expect it to be a flavoenzyme. If it really is a flavoenzyme, there might be a tiny amount of FAD - too little to see - bound to your preparations. Thus your sample may have low residual activity detectable by your assay, but the specific activity would be very low.
CD is not likely to be helpful here; it could be useful for other applications. CD is much less sensitive than absorbance spectroscopy, so it's only really useful when most of the enzyme has flavin bound, i.e., after you solve your immediate problem. We've collected CD spectra from 300 nm - 700 nm of a few dozen oxidized flavoproteins and they vary wildly; sometimes the signals are strong, sometimes not.
As for a "U-shaped conformation" of FAD... That's an appropriate description for a few flavoenzyme structures, such as DNA photolyase. And FAD folds over on itself in aqueous solution due to the hydrophobic interactions between the isoalloxazine and adenine ring systems. In most cases, however, FAD bound to proteins adopts a relatively extended conformation, with the isoalloxazine and adenine ring systems being quite distant.
Hi! check activity of your protein with and without addition of FAD. Activity level shows your protein cofactor dependence. If you have purified protein , then go for dialysis. check your protein activity after dialysis, activity will decrease if protein is actually cofactor dependent. During dialysis, usually cofactors will leak out of your dialysis membrane.
There are some theoretical studies about it considering the sequence. See for instance Protein Sci. 2001 September; 10(9): 1712–1728. PMCID: PMC2253189. Sequence-structure analysis of FAD-containing proteins.Orly Dym and David Eisenberg. I remember some others published more recently. It will be easy to find out. Good luck
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