Hi Yvonne, Were you able to check the polydispersity of your highly soluble protein sample by DLS? Non-monodispersity could result in reduced propensity for crystallization. Another thing you could then try is something like the "Optimum Solubility Screen" which basically involves buffer exchanges into different pH conditions to find a condition which is monodisperse. You can refer to the following paper for details:
Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug 2. Jancarik J, Pufan R, Hong C, Kim SH, Kim R. More recently, similar approach has been also reported in: Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Acta Crystallograph Sect F Struct Biol Cryst Commun. 2005 Dec 1;61(Pt 12):1035-8. Epub 2005 Nov 5. Collins B, Stevens RC, Page R. Also, other than reductive methylation and surface mutagenesis, you can also consider protein truncation based on structure prediction progams and homology modeling. Regards, Debanu. -- Debanu Das, JCSG, SSRL. ----------------- -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Stephen Graham Sent: Tuesday, August 07, 2007 11:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] highly soluble proteins Reductive methylation of amino groups (lysines and the N-terminus) is a fairly routine chemical modification that should drop the solubility of your protein. An easy-to-use protocol can be found in Walter et al (2007) "Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization." Structure, 14:1617-1622 Cheers, Stephen On 8/7/07, [EMAIL PROTECTED] <[EMAIL PROTECTED]> wrote: > Yvonne, > > Several 'old' proteins have been crystallized from insanely high > concentraitons - concanavalin A for instance can be grown from 100-250 > mg/ml solutions by means of 'salting in' using microdialysis. This is > of course highly labor-intensive and also expensive on the protein side. > > Look at the distribution of charged residues on the protein > (especially helpful if you have a model of some sort). I would > recommend mutating some of them to non-charged or even nonpolar > residues in order to lower solubility. Alternatively, you could try > preparing chemical derivatives of the protein using e.g. heavy atoms or > amine-modifying reagents like NHS. > If you block enough amines, your solubility should go down and your pI > will change as well. Likewise you could try modifying exposed acidic > residues etc. > > Mutagenesis can in the end be easier to do because chemical > modification tends to produce complex mixtures of products, unless you > do it with a huge excess of the reagent and allow the reaction to > proceed to exhaustion. > > Artem > > > Our lab is trying to crystallize a highly soluble (100+ mg/ml) > > protein with a molecular weight of 35 kd. > > > > The protein was screened against 1536 conditions at 20 mg/mL. Most > > drops were either clear or produced "bubbles" (often oily looking). > > The few that had precipitate contained high concentrations of K3PO4, > > cobalt, or zinc. We have tried repeating some of the bubble > > conditions at 100+ mg/mL and are still getting clear drops or bubbles. > > > > Is there something about highly soluble proteins and/or secreted > > proteins and/or proteins with unusual portions of their sequence > > that needs to be considered in order to successfully crystallize it? > > > > I am considering trying "salting out" using dialysis, and also > > adding ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer. > > > > I welcome thoughts and suggestions on crystallization ideas, > > publications, etc. > > > > Thank you > > > > Yvonne > > > -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
