*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk ***
Most of halophilic proteins are highly charged; you might like to check their crystallisation conditions in the pdb. For example, Hm MalDH, a halophilic protein (pI 4.5), was crystallised by reverse vapour diffusion from high salt in presence of MPD. See: Costenaro, L., Zaccai, G., and Ebel, C. (2001). Understanding the crystallisation of an acidic protein by dilution in the ternary NaCl - 2-methyl-2,4-pentanediol - H2O system. J. Crystal Growth 232, 102-113. http://dx.doi.org/10.1016/S0022-0248(01)01150-2 The driving force behind is depletion of MPD molecules around the charged surface of the protein, when the salt concentration and screening decrease. Regards, ___________________________________ Dr. Lionel Costenaro Institut de Biologia Molecular de Barcelona - CSIC Parc Cientific de Barcelona Josep Samitier 1-5 08028 Barcelona, Spain Phone +34 93 403 49 57 Fax +34 93 403 49 79 Email [EMAIL PROTECTED] URL www.ibmb.csic.es -----Mensaje original----- De: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] En nombre de Kornelius Zeth Enviado el: jueves, 11 de enero de 2007 18:11 Para: ccp4bb@dl.ac.uk Asunto: [ccp4bb]: Off topic: Crystallization of highly charged protein *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Dear all, we are trying to crystallize a protein (40 kD) that was cloned de novo as construct of five identical repeat structures. The protein contains no LYS, ARG, GLN, GLU, CYS, MET but a large number of negative charges (pI 3.7). The protein appears properly folded from CD, size exclusion. Crystallization hasn't worked out at 10 - 50 mg/ml. We also tried 10 - 50 mg/ml in presence of Ca and Zn, with and without Histag, we also added small diamines. Nothing worked so far. Recloning and mutational changes are not easy due the repetitive gene structure, however they are possible as the last anchor (including gene synthesis). 1. As we are very much interested in the protein structure I wonder whether people from other labs have any experience with highly charged proteins? 2. Are there any proteins with a significant surplus of positive charges crystallized that might be co-crystallized? 3. Did people use any chemicals to modify ASPs or GLUs side chains? Best wishes and thanks for your help! Kornelius ---------------------------------------------- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
