Hi Michael Offering a different way to look at it:
There is an energy cost to de-solvate a precipitant molecule, and vice-versa. Crystallization allows protein to bury surface which is normally solvated, freeing waters to the "bulk" solvent. These water molecules are then available to solvate the precipitant. Hence the packing process becomes favorable in spite of the entropy cost. If there is an energy gradient then there is a driving "force" to crystallization. But we may agree albeit for a nomenclature issue. As for "all protein being packable", the supporting data are incomplete to prove it one way or the other. It is an experimental fact that some proteins are "uncrystallizable" within the available crystallization space attempted by a scientist. Yet these proteins may be monodisperse, in homogenous form as required for crystallization. I have partial - and unpublished - evidence that "packability" may play a role. It involves a series of experiments which affect the shape of a protein by addition of various ligands. Depending on whether a projected loop collides with a crystal contact the complex with a ligand becomes "crystallizable" (trivially, just using APO conditions)or not (even by limited broad screening). Soaks allow to see where the loop projects except that its end becomes disordered when it is projected towards the crystal contact. This is a system for which several groups have published different crystal conditions and yet only one single space group has ever been found. Deprive that protein from being able to form the APO packing and it becomes at the very least much more difficult to crystallize. Thierry From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of R. M. Garavito Sent: Wednesday, April 08, 2015 11:27 AM To: [email protected] Subject: Re: [ccp4bb] Crystallization of a minority fraction monomers Thierry, I need to point out there is no outside work as it is one system, but with multiple phases. Protein and nucleic acids are not true crystals in the classic sense, but highly hydrated ordered colloids (in the 1930's some called them "crystalloids" because bulk water is such a major and critical component, unlike small molecule crystals). It is colloidal physical chemistry at work. Thus, the water argument for a "force" does not hold, rather the system just comes to an energy minimum where two stable phases are formed (one being the crystal). My complaint is that we use terms that imply the wrong physical behavior, which then obscure the true issues. For example, every protein is packable from a purely physical standpoint; physical shape is not the issue, but the balancing of favorable and unfavorable interactions is. Crystallization is a balance between many global and local interactions. Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected]<mailto:[email protected]> **************************************************************** On Apr 8, 2015, at 10:52 AM, "Fischmann, Thierry" <[email protected]<mailto:[email protected]>> wrote: Some counter-arguments to Michael : There is an "outside force doing the work": macromolecule crystallization except rare exceptions is driven by competition for water molecules between the macromolecule and the precipitant. The exceptions are crystallization against low salt buffer, in which case the process is driven by hydrophobic "forces". And "packable" may play a role. A molecule which is of such shape and surface charge distribution that there is no way to pack it in a regular lattice will never crystallize. Regarding the dimer vs. monomer debate, crystallization acts as a purification step. It seems perfectly plausible that crystal growth would "select" the monomeric state if dimers cannot be included in the growing crystal lattice, regardless of whether one is more soluble than the other. It all comes down to the initial crystal seed favored by the crystallization conditions. On a separate note, protein which forms dimers in solution trend to be more soluble in dimeric state than as monomers because dimerization usually buries a significant hydrophobic patch of molecular surface. If crystallization was only "selecting for the least soluble" oligomeric state we would rarely crystallize proteins as dimers. Crystallization is such a confusing process :) Thierry From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of R. M. Garavito Sent: Wednesday, April 08, 2015 10:04 AM To: [email protected]<mailto:[email protected]> Subject: Re: [ccp4bb] Crystallisation of a minority fraction monomers I just wanted to disagree with Roger's word choice, but not his argument (this is a "flame"-free response). Forget about "packing" and "packable" as there is no outside force doing the work. The molecules are just falling into a local energy minimum where favorable intra- and intermolecular interactions predominate. It is difference in the behavior of the ensemble versus of a solubilized, dispersed species (be it monomer or dimer). It is a phase behavior issue. Concerning Sebastian's case, while it is uncommon, the idea that a monomer has a crystalline phase state while the dimer does not is perfectly reasonable, and the crystals of the monomer grow due to mass action. I am sure the number of verified examples of this are limited. However, there are many cases where dimeric and tetrameric enzymes can be shown to be fully saturated with one or another bound substrate in solution, but show one or more empty active sites in the crystal. I know of several cases where this occurs, showing that selection of the species with the best set of favorable intra- and intermolecular interactions occurs. Regards, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected]<mailto:[email protected]> **************************************************************** On Apr 8, 2015, at 9:28 AM, Roger Rowlett <[email protected]<mailto:[email protected]>> wrote: The problem with crystallization is that is selects for the least soluble, most packable species. Sometimes that works against what you would like to know. That could include oligomerization state as well as conformational state. For example, some of the allosteric carbonic anhydrases stubbornly crystallize only in the T-state, despite crystallization conditions that are known to preferentially stabilize the R-state, and for which the predominant R-state population can be confirmed by other methods. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [email protected]<mailto:[email protected]> On 4/8/2015 9:07 AM, Sebastiaan Werten wrote: Dear all, we are currently working on a protein that is known to exist in a monomer-dimer equilibrium. At the high concentrations used for crystallisation assays, the dimer is predominant and the monomer practically undetectable. Nevertheless, one of the crystal forms that we have obtained contains the monomeric species, not the dimer. I was wondering if anyone is aware of similar (published) cases, and if the phenomenon as such has been discussed in detail anywhere? I did literature searches but so far couldn't find anything. Any pointers would be much appreciated! Best wishes, Sebastiaan Werten. Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (2000 Galloping Hill Road, Kenilworth, New Jersey, USA 07033), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (2000 Galloping Hill Road, Kenilworth, New Jersey, USA 07033), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
