surface residue mutation

From: Prem Kaushal
Date: 14 February 2012 23:36

Hi

We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me.

Thanks in advance

Prem

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From: Bernhard Rupp (Hofkristallrat a.D.)

 

http://services.mbi.ucla.edu/SER/

 

but no space group predictions are possible. BR

 

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From: Antony Oliver

Have you solved the structure? It's just that you don't say why you need different crystal forms. 

We had to do a bit of "crystal engineering" in order to get a complex between our protein and a peptide. It turned out to be relatively simple case; visually inspecting the crystal packing (in Coot) then mutating, in our case a single amino acid, that was generating the crystal lattice. 

If you have the structure you could use PISA from the EBI to look for your lattice contacts, and choose amino acids there. 

If you don't have a structure then things are obviously a bit more complicated. You could try and generate homology models (Phyre2) and then mutate surface residues, either obvious hydrophobics predicted to point out to solvent, or make charge reversal mutants (K to E and vice versa). 

Wishing you good luck,

Tony. 

Sent from my iPhone

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From: <alexander.pautsch

Hi Prem,

besides trying surface entropy reduction you can also start by analyzing your crystal contacts and mutate residues therein. This was for example investigated in the 90's by GE Schulz and coworkers.

Good Luck

Alex

 

 

 

 

 

Dr. Alexander Pautsch 

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From: Enrico Stura


Dear All,

One of the most efficient methods to change space group and packing without having to change
the sequence is to change the length of N and/or C terminal tags.

An example that I am familiar with is given by the following PDB codes.

1JIZ, 1RMZ, 1JK3,  1UTT, 1UTZ, 2WOA, 2W0D, 1ROS, 1OS9, 3BA0

It includes 1 surface residue mutation, but the rest are small variations in length.

Complexation with any ligand that may protrude is also likely to work.

Enrico.

Enrico A. Stura D.Phil.

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From: David Schuller

Wukovitz & Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067
predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly.

--  ======================================================================= All Things Serve the Beam =======================================================================                                David J. Schuller

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From: Jacob Keller


Are there any all-D proteins out there, of known structure or
otherwise? If so, do enantiomer-specific catalyses become inverted?

JPK

--
*******************************************
Jacob Pearson Keller

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From: Kelly Daughtry

The most famous case I know of was the HIV protease. My grad school PI used to use it as an example in class.

Science. 1992 Jun 5;256(5062):1445-8.

Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected].

Milton RC, Milton SC, Kent SB.

http://www.ncbi.nlm.nih.gov/pubmed/1604320 

Kelly
*******************************************************
Kelly Daughtry, Ph.D.

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From: Chris Lemke

Building on Alex's suggestion, here are some papers on rationally engineering new crystal contacts:

 

via disulfides:

http://www.pnas.org/content/103/44/16230.long

http://onlinelibrary.wiley.com/doi/10.1002/pro.550/abstract;jsessionid=783A91CC93571B0348F37A325929676D.d01t02

 

or via  leucine zippers:

http://onlinelibrary.wiley.com/doi/10.1110/ps.072851407/abstract

 

Good luck,

Chris.

 

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From: David Schuller

On 02/15/12 12:41, Jacob Keller wrote:

Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted?  JPK 

What do you mean by "Out There"? If you mean in the PDB, then yes.  As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4  C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues.

Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide.

On the other hand, if by "out there" you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis.

The first paper I managed to Google:
http://jb.asm.org/content/185/24/7036.full
Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides
doi: 10.1128/​JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043

If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization.

On Wed, Feb 15, 2012 at 8:05 AM, David Schuller > wrote: 

Wukovitz & Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly.   On 02/14/12 18:36, Prem Kaushal wrote:   Hi  We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me.  Thanks in advance  Prem  

--  ======================================================================= All Things Serve the Beam =======================================================================                                David J. Schuller 

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From: David Schuller

 On 02/15/12 12:41, Jacob Keller wrote:

Are there any all-D proteins out there, of known structure or
otherwise? If so, do enantiomer-specific catalyses become inverted?

JPK

I looked a little harder, and at least one D-enantiomeric protein was an enzyme:

Total chemical synthesis of a D-enzyme: the enatiomers of HIV-1 protease show demonstration of reciprocal chiral substrate specificty
R.C. deL. Milton, S.C.F. Milton, S.B.H. Kent (1992) Science 256(5062) 1445-1448.

I guess that answers your question.

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From: Jacob Keller

Right on the money!

JPK

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From: Joel Tyndall


Steve Kent has published a few more (at least 1 other) since HIV...

3ODV http://www.rcsb.org/pdb/explore/explore.do?structureId=3ODV

Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography.

Pentelute, B.L.,  Mandal, K.,  Gates, Z.P.,  Sawaya, M.R.,  Yeates, T.O.,  Kent, S.B.,

Journal: (2010) Chem.Commun.(Camb.) 46: 8174-8176

_________________________________
Joel Tyndall, PhD

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From: Prem Kaushal

Dear all,

Thanks for your response to my quarry about surface residue mutation. I tried the SERp Server, but did not get satisfactory clue. This software classified our protein as difficult to crystallize, however we have crystallized and solved the structure also. But our crystal form is not good for soaking experiments. Wondering is there any other software.  

Thanks

Prem

--
Prem S. Kaushal