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We had a similar situation with a catalytically dead serine protease.  Initially I was excited to think we might be seeing residual catalytic activity of the mutant enzyme on a highly specific substrate; however, the activity turned out to result from contamination with a very small amount of wt enzyme, likely as a result of using the same affinity column to purify both.  When we incubated the enzyme preparation with PMSF (an inhibitor that covalently modifies the catalytic serine and would not have affected the mutant), we eliminated the residual activity of the enzyme preparation.  However, in our case we never were able to get crystals with the intact substrate, which apparently was not compatible with our crystal form. 

 

Is there a covalent inhibitor of your cysteine protease that you could use to pre-treat your enzyme, to see if this eliminates the activity?  If so this might help distinguish between residual activity of the mutant vs. contamination with wt enzyme.

 

Good luck!

Evette

 

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant

Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372

http://www.mayo.edu/research/labs/proteases-cancer/

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Dipankar Manna
Sent: Monday, April 20, 2015 2:42 PM
To: [log in to unmask]
Subject: [ccp4bb] Cleaved peptide density!

 

Dear Crystallographers,

 

I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how!

 

Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). 

 

If anybody has the same kind of experience please advice me.

 

Thanks in advance.

 

Best,

 

Dipankar

 

--

Dipankar Manna

Research Scholar

Department of Chemistry

University of Oslo

Oslo, Norway