[ccp4bb] Crystallization of lysine and arginine rich proteins
Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Try reductive methylation of the lysines: Walter, T. S., Meier, C., Assenberg, R., Au, K. F., Ren, J., Verma, A., Nettleship, J. E., Owens, R. J., Stuart, D. I. Grimes, J. M. (2006). Structure, 14, 1617–1622. Cheers, Stephen 2009/11/2 Jan Rash jan...@googlemail.com: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Hi Jan, We have tried crystallizing a similar protein without success, later on it turned out that the protein was having a strong interaction with DNA that was not sequence specific. You might have a case like that Flip Jan Rash wrote: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Following on from Stephen's comment. Jena Biosciences sell a kit that allows you to do it quickly and easily. check it out here: http://www.jenabioscience.com/cms/en/1/catalog/529_jbs_methylation_kit.html We have had success in our lab using lysine methylation to crystallise an otherwise recalcitrant protein. Good luck Simon Stephen Graham wrote: Try reductive methylation of the lysines: Walter, T. S., Meier, C., Assenberg, R., Au, K. F., Ren, J., Verma, A., Nettleship, J. E., Owens, R. J., Stuart, D. I. Grimes, J. M. (2006). Structure, 14, 1617–1622. Cheers, Stephen 2009/11/2 Jan Rash jan...@googlemail.com: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Dear Umar, It's probably not local flexibility or the interaction with other molecules, such as DNA, that prevents your protein from crystallising but an unfavourable enthalpy/entropy balance of long side chains which are potentially restricted by forming crystal contacts. Mutation of these to small, low-entropy residues such as alanine might help. Have a look at the surface entropy reduction prediction server for example (http://nihserver.mbi.ucla.edu/SER/intro.php) and see if this could be helpful in your case. Cheers and good luck, Uli Gohlke --- dr ulrich gohlke staff scientist - macromolecular structure and interaction max-delbrück-centre for molecular medicine (mdc) +49 30 9406 - 2725 (w) +49 30 9406 - 2548 (fax) ulrich.goh...@mdc-berlin.de http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jan Rash Sent: Monday, November 02, 2009 2:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystallization of lysine and arginine rich proteins Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Umar, Check out: Czepas et al. The impact of Lys--Arg surface mutations on the crystallization of of the globular domain of RhoGDI, Acta D (2004) 60 275-280. They point out that sulfate ions can help mediate contacts between arginine residues from neighboring molecules in the crystal. You may have already considered the surface entropy server (http://nihserver.mbi.ucla.edu/SER/) to help identify any specific stretches of amino acids that could be mutated to reduce entropy and possibly promote crystallization. Maybe there are a couple regions of your sequence that are flagged as particularly unfavorable in terms of their predicted entropy contribution. There is evidence that lysine is more of a problem in this respect as compared to arginine (Derewenda et al. Acta D (2006) 62 116-124). Good luck, -Andy === Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853 === On 11/2/2009 8:39 AM, Jan Rash wrote: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Before deciding to change the protein sequence it is best to look at the precipitation pattern of the protein as a function of various precipitants. If this shows that precipitation is not within the standard range commonly observed for other basic proteins even at high protein concentrations then side chain mutation should be tried. Even lysozyme would be hard to crystallize at 1 mg/ml at 38°C. E. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab LTMB, SIMOPRO, IBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette Cedex FRANCE http://www-dsv.cea.fr/en/ibitecs/82 http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
