If you have an assay for the activity of your peptidase, you can check which metal ion is best able to reconstitute the activity of your protease. Most bacterial metalloproteases use zinc, often bound through a HEXXH motif (Article Metalloproteases

Article Metal Preferences of Zinc-Binding Motif on Metalloproteases

Dear Davis

First of all, generally looking for conserved domain in your protein (performing blast, smart at EMBL or other tools) you can try to identify the metal biding site.

As Annemarie suggest, fos example the presence of a HEXXH motif or similar can suggest that your protein is a zinc protease.

However you can try to check it experimentally in some way:

1) Set-up an in vitro activity assay. For example incubate your protein with different metal and check if you can detect is activity using a protease activity assay (eg. https://www.thermofisher.com/order/catalog/product/E6638 or https://www.thermofisher.com/order/catalog/product/E33758) or if the substrate of your protease is it known and available commercially just incubation the protease with and with/out metal with the substrate and check the protease activity via SDS-page (if the digestion is detectable). Of course this experiment need to be carefully designed.

2) In case you are not able to set-up an in vitro activity assay because you do not know the substrate of your protease, other option could be compare the ability of different metal s to binding to the protein, eg.:

a) monitoring the shift in melting temperature of the protein titrated with different metals by DSF (differenzial scanning fluorimetry) that if you have access to a RT-PCR instrument is very simple assay to set-up

b) monitoring the chemical shift thats happen in a 15N HSQC spectra (if you are using NMR to solve your structure)

Of course those last approaches are indicative and an activiy assay is preferable.

good luck

Manuele

I agree with Annamarie, chelate with EDTA so there is no activity and then add back different metals to see which restores activity.

You could submit a sample of your protein for atomic absorption spectroscopy or ICP-MS. It is crucial to eliminate background metal contamination prior to these procedures.

submit your purified protein for metal analysis. You might be lucky enough to find a device capable of doing it near your lab. atomic absorbance by flame photometry is not good enough you will need it to be equipped with a graphite furnace. MP-AES (microwave-plasma atomic emission spec) is very good but the best would be ICP-AES or ICP-MS.

Chemists however (where you find most machines like this) will tell you your sample is too dirty for their machine (as its organic material that they fear will clog it up).

Unfortunately you must sacrifice some of your protein just to be sure of the metal content. I am surprised by how many enzymologists do not do this and assume that their protein (even active site mutants) contain the 'correct' metal.

There are companies that will do it for you if you can afford it.

NO! don't try to predict what your protein contains in terms of metal content. You can only predict sites that might bind metals. What is ACTUALLY in the protein cannot be predicted. You might make an educated guess using software but do not assume it is true or real.

Only direct analysis will tell you whats in there.