[ccp4bb] SUMMARY about deblock N-terminus

- Protein crystallography

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- Protein purification
- Crystallisation

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: An off-topic question about mitochondria protein expression
From: "Yong, Wei" WYong {- at -} MCW {- dot -} EDU
Date: 2007-11-19

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Subject: SUMMARY about deblock N-terminus
From: "Yong, Wei" WYong {- at -} MCW {- dot -} EDU
Date: 2007-11-19

Dear friends,

Thank you very much for your suggestions which are very helpful. I am writing to thank you from the bottom of my heart. Here is the summary of the topic.

Best

Wei
#########################################

QUESTION:
I solved a protein crystal structure which is about 95KD and was purified from pig liver. Based on the crystal structure, I missed 36 residues at its N-terminus and 16 at its C-terminus. I tried to do N-terminus sequencing to identify if these 36 residues was cleaved or not. However, the N-terminus sequencing failed. One of the possibilities is that the N-terminus has been blocked by some post-translational modifications, such as acetylation and methylation, which leave no free NH2 group. Can anybody give me some suggestions about how I can deblock the modifications so that I can do N-terminus sequencing? Or any other suggestions on how to identify from which residue my protein starts (Other techniques).

*******************************
Jinghua from NIH/NIAID wrote:
You may want to do mass spec. to get the exact molecular weight of your
protein.
In addition, the mass spec for protein Identification can give you the
sequence of your protein. You can check the details with the facility in
your University.
*******************************
Bernhard Rupp wrote:
Mass spec of the crystal
*******************************
Roopa Thapar wrote
If you have access to a mass spec/proteomics facility, it should be easy
to get an intact mass on your protein followed by N-terminal sequencing
using MS/MS.
If your sequence begins with a Met-Ser, then it is likely that the
N-terminal Met is cleaved and the Ser is acetylated.
*******************************
Wei Qiu wrote
It's quite normal that some residues at N- or C-terminal are missing in the final model since they are disordered in the crystal. If you really want to know what's in your crystal, you can pick up one big crystal, wash carefully with buffer (to get rid of the precipitant, such as PEG) and run Mass Spec for the molecular weight. I bet you probably will get a MW corresponding to the full length protein.
Or you can try 8M urea to unfold your protein and do sequencing again.
***********
Petra wrote
could as well be that your first residue is Gln which frequently cylizes
to pyroglutamate which resists Edman chemistry. In that case you can
deblock your N-terminus with pyroglutamate aminopeptidase (you can get it
from Takara, Sigma, Boehringer, Pierce - whicheverone you like ;-)). THere
are well working standard protocols.
***************
Remy Lorisµ wrote:
Mass-spec should give you the corect molecular weight and tell you if the protein is full length or truncated. It is not uncommon to have N and/or C-termini disorderedand invisible in crystal strutures though. This is not something to be worried about
*******************
Dr JJ Kim wrote
why don't you calculate the theoretical MW of SD including the
natural abundance of C, O, N and H (of course, deuterium) and see
whether you can distinguish the difference between the full length (
mature protein) and the truncated protein. also, if indeed you have
the extra ~30 residues in your structure, is htere room for them in
the crystal lattice?

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: An off-topic question about mitochondria protein expression
From: "Yong, Wei" WYong {- at -} MCW {- dot -} EDU
Date: 2007-11-19

Next message:
Subject: Re: Unmerged output from Scala
From: Clemens Vonrhein vonrhein {- at -} GLOBALPHASING {- dot -} COM
Date: 2007-11-19