From: Chaudhary, Ritcha
Date: 6 September 2011 17:51
Dear all
I am interested in crystallizing a 5 residue peptide. I have no prior experience in this field although I have crystallized larger proteins (5--60 kDa). Do people use regular screens used in macromolecular crystallization such as Hamptons, wizards etc? Any suggestions are greatly appreciated. Also, can anyone point me to some relevant literature ? Thanks.
Ritcha
----------
From: Ed Pozharski
Probably not - I believe short peptides are crystallized mostly via
various evaporation techniques (but I am very curious to hear what those
with more experience with this will suggest). It should also form a
stable structure, otherwise it will either not crystallize or the form
you get won't be representative of solution structure.
Which begs the question - why not NMR?
--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs
----------
From: Mark J van Raaij
Dear Ritcha,
we have crystallised various peptides, especially circular ones, both from regular screens used in MX and from specially prepared screens (i.e. different organic solvents).
We tend to use slow evaporation from under mineral oil in Terazaki plates (i.e. microbatch).
We have also failed to crystallise many peptides, perhaps due to their inherent flexibility, perhaps due to our incompetence. I also did not have previous experience in crystallising peptides.
What to try first depends on your peptide. If you have a lot of material, you can just try many, many conditions.
If you have it as a solid or can obtain it as a solid by evaporation, you can quickly determine solubility in different solvents. We find 50% methanol useful in many cases. If you try volatile solvents first, you can then evaporate them and re-use the peptide.
From your sequence, you should know if your peptide is hydrophilic, hydrophobic, aromatic, basic, acidic, etc. Based on these properties dissolve at high concentration in "like" solvent and set up crystallisation trials with solutions likely to precipitate it out "slowly", i.e. somewhat "unlike".
If your peptide is very hydrophobic, you may need to use hydrophobic solvent incompatible with normal plastics. In these cases we use glass containers.
If you have organic chemists nearby, talk to them - many of them have experience in crystallising organic compounds and can give you good ideas for crystallising peptides.
Greetings,
Mark
Mark J van Raaij
Date: 6 September 2011 17:51
Dear all
I am interested in crystallizing a 5 residue peptide. I have no prior experience in this field although I have crystallized larger proteins (5--60 kDa). Do people use regular screens used in macromolecular crystallization such as Hamptons, wizards etc? Any suggestions are greatly appreciated. Also, can anyone point me to some relevant literature ? Thanks.
Ritcha
----------
From: Ed Pozharski
Probably not - I believe short peptides are crystallized mostly via
various evaporation techniques (but I am very curious to hear what those
with more experience with this will suggest). It should also form a
stable structure, otherwise it will either not crystallize or the form
you get won't be representative of solution structure.
Which begs the question - why not NMR?
--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs
----------
From: Mark J van Raaij
Dear Ritcha,
we have crystallised various peptides, especially circular ones, both from regular screens used in MX and from specially prepared screens (i.e. different organic solvents).
We tend to use slow evaporation from under mineral oil in Terazaki plates (i.e. microbatch).
We have also failed to crystallise many peptides, perhaps due to their inherent flexibility, perhaps due to our incompetence. I also did not have previous experience in crystallising peptides.
What to try first depends on your peptide. If you have a lot of material, you can just try many, many conditions.
If you have it as a solid or can obtain it as a solid by evaporation, you can quickly determine solubility in different solvents. We find 50% methanol useful in many cases. If you try volatile solvents first, you can then evaporate them and re-use the peptide.
From your sequence, you should know if your peptide is hydrophilic, hydrophobic, aromatic, basic, acidic, etc. Based on these properties dissolve at high concentration in "like" solvent and set up crystallisation trials with solutions likely to precipitate it out "slowly", i.e. somewhat "unlike".
If your peptide is very hydrophobic, you may need to use hydrophobic solvent incompatible with normal plastics. In these cases we use glass containers.
If you have organic chemists nearby, talk to them - many of them have experience in crystallising organic compounds and can give you good ideas for crystallising peptides.
Greetings,
Mark
Mark J van Raaij
No comments:
Post a Comment