Are you talking about the respective ligand with progesterone or estrogen receptor ligand binding domains? That has been done many times. Larger pieces of the receptor have proven more difficult.
Adding 10uM compound in the fermentation media is the traditional route, because a significant portion of the receptor misfolds in bacteria otherwise. If you do want to add compounds to concentrated protein, don't worry about compound solubility. We have solved over 20 ER LBD structures with different compounds by diluting 100mM stocks in 100% ethanol or DMSO to 1mM. Most of the compound crashes out, but gets soaked up by the protein. The next day we microcentrifuge and set up trials with supernatant. We used to carbamylate free cysteines with iodoacetic acid, but switched to high MBE (10-50mM), which gives adducts. You'll also want the LxxLL peptide at 3-5 fold excess. With ER LBD, almost all our agonist conformation structures are in PEG3350. Also, we never did get a structure with estradiol; the receptor crystallized readily with genistein. See this paper for purification details. http://www.nature.com/nchembio/journal/v4/n4/abs/nchembio.76.html Regards, Kendall. On 4/1/09 12:59 PM, "KUMARASWAMI MUTHIAH" <megun...@hotmail.com> wrote: > Anybody tried to cocrystallize the protein-progesterone or estrogen complexes, > if so how do you go about the solubility of these compounds? Progesterone is > only soluble in 50% chloroform or 100% DMSO and the dilution of this stock is > not possible as chloroform falls out of solution. Lots of papers out there > used soaking with progesterone or expressed the protein in the presence of > progesterone. Any suggestions would be appreciated. > Thanks > > > > > Rediscover Hotmail®: Now available on your iPhone or BlackBerry Check it out. > <http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobi > le1_042009>
