Re: [ccp4bb] Solvent content of membrane protein crystals
Das, Debanu wrote: Hi, There are at least 4 methods to try to estimate amount of detergent in a membrane protein crystal . In summary, someone wanting to estimate amount of detergent in their crystals and have sufficiently large and numerous crystals, could try out any of the above methods. The above techniques are quite well documented in literature. The first 3 can be done in-house and so can FTIR. I happen to have the reference for Ron Kaplan's TLC method at my fingertips: Eriks, L. R., Mayor, J. A. Kaplan, R. S. (2003). A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins. Anal Biochem 323, 234-41. Sensitivity is limited by the amount of aqueous solution you can spot on a TLC plate. Probably could improve sensitivity by speed-vac-ing an aliquot to near dryness and redissolving in 50% methanol or something.
Re: [ccp4bb] Solvent content of membrane protein crystals
Quoting Edward Berry [EMAIL PROTECTED]: Savvas Savvides wrote: Indeed, but wouldn't consideration of micelle size affect our estimation of the number of molecules in the asu, in some cases significantly? Good point- I think now that is taken into account by just saying membrane proteins tend to have a high solvent content and taking that into consideration when you guess the number of molecules. But it would be nice to account for the detergent explicitly. Say by analyzing detergent content of the crystals, or in some ideal cases neutron diffraction with perdeuterated detergent. with regard to this has anyone actually checked how the micelle properties with or without protein embedded might differ?? are we assuming empty micelle and the protein-added micelle are the same size/Mw? is this really so? --- of course this may further vary depending on the oligomeric state of the protein --suppose some neutron scattering studies on model systems might give the answer --havent looked. just wondering.. -tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Solvent content of membrane protein crystals
Saavas and Tommi, The questions of what is the detergent content of a membrane protein crystal and how to explicitly determine the amount of detergent in a crystal are extremely difficult to address. Moreover, is it worthwhile to even attempt to correct the Matthews coefficient? I personally don't for a number of reasons. However, one point I would like to make in this discussion is that ANYTHING concerning micellar structure or behavior cannot be naively extrapolated to a protein- detergent complex without firm experimental data. Moreover, when the protein-detergent complex is in a crystal, it gets even worse. Very little quantitative work has been done on what is the detergent structure and behavior in a protein-detergent complex. Peter Timmins has done the most using neutron diffraction with me and Wolfram Welte on crystalline systems, as well as in solution (one paper is below). Pebay-Peyuola, E., Garavito, R.M., Rosenbusch, J.P., Zulauf, M., and Timmins, P.A. (1995) Detergent structure in tetragonal crystals of porin from the outer membrane of E. coli. Structure 3, 1051-1059. One immediate take home message is that a membrane protein IS NOT in a micelle, even by definition from surfactant chemistry, nor does a membrane protein insert into a micelle. In many of the experiments on detergent binding in surfactant chemistry using styrene beads, detergent adsorbs onto a hydrophobic surface from single monomer accretion, and perhaps by micelle fusion. Hence, one should forget about micelles when talking about a protein-detergent complex. My rule of thumb from experience is that an average membrane protein of about 50 KD binds about a micelle's worth of detergent, but it would be a mistake to assume it has all the characteristics of a free and pure detergent micelle. Getting back to the amount of detergent in a crystal and the Matthews coefficient, the detergent layer of protein-detergent complex can behave like a hard sphere in a crystal or it can fuse with its neighbors, depending on the detergent used. Changing the detergent concentration around the crystal, as we do when manipulating a crystal for many experiments, will change the detergent concentration in the crystal and can impact the detergent layer of protein- detergent complex. Thus, efforts to get accurate, detergent- corrected Matthews coefficients for membrane proteins, may not be worth worrying about. Regards, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Sep 24, 2007, at 2:29 AM, Tommi Kajander wrote: Quoting Edward Berry [EMAIL PROTECTED]: Savvas Savvides wrote: Indeed, but wouldn't consideration of micelle size affect our estimation of the number of molecules in the asu, in some cases significantly? Good point- I think now that is taken into account by just saying membrane proteins tend to have a high solvent content and taking that into consideration when you guess the number of molecules. But it would be nice to account for the detergent explicitly. Say by analyzing detergent content of the crystals, or in some ideal cases neutron diffraction with perdeuterated detergent. with regard to this has anyone actually checked how the micelle properties with or without protein embedded might differ?? are we assuming empty micelle and the protein-added micelle are the same size/Mw? is this really so? --- of course this may further vary depending on the oligomeric state of the protein --suppose some neutron scattering studies on model systems might give the answer --havent looked. just wondering.. -tommi -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] Solvent content of membrane protein crystals
Savvas Savvides wrote: Indeed, but wouldn't consideration of micelle size affect our estimation of the number of molecules in the asu, in some cases significantly? Good point- I think now that is taken into account by just saying membrane proteins tend to have a high solvent content and taking that into consideration when you guess the number of molecules. But it would be nice to account for the detergent explicitly. Say by analyzing detergent content of the crystals, or in some ideal cases neutron diffraction with perdeuterated detergent. The crystal packing of some membrane proteins shows that they tend to pack as potatoes in space with relatively few protein-protein contacts and with detergent micelles presumably providing the rest of the crystal packing interactions. That also explains the often significant diffraction anisotropy observed in such crystals. One classic example is the prototypical potassium channel structure (KCSA) (PDB entry 1bl8). I'll have to look at KCSA again. I've been assuming the micelle is too fluid and solvent-like to make any kind of a crystal contact, but it occupies space holding the molecules apart and preventing real crystal contacts. This was the rationale behind Michel's use of small amphiphiles to replace the bulky micelle, and antibody fragments to bridge the gap and provide hydrophilic areas for contact. Savvas Quoting Edward Berry [EMAIL PROTECTED]: I would use a very general definition for solvent, including disordered detergent and lipids. As you know in many cases ordered detergents and lipids have been modeled in the coordinates, so they are part of the model not the solvent. In some cases I think waters should be included in the model not solvent- say for structural waters buried in the protein at least. Ed Savvas Savvides wrote: Dear colleagues, in estimating the solvent content of membrane protein crystals it would only seem reasonable that micelle size should also be taken into account. Depending on the aggregation number and MW of a given detergent, the concentation of detergent used, and the buffer conditions, one may have micelles on the order of 15-25 kDa or even 35-50 kDa for detergents with alkyl chains of more than 10 carbons. However, when I took a look in a handful of papers reporting Matthews' numbers for membrane protein crystals, it became apparent that only the protein MW is used in such estimates. I am beginning to wonder if one should even bother reporting a Matthews number for a membrane protein crystal given the uncertainties surrounding size and role of micelles in crystal packing. Any thoughts on this? best wishes Savvas
[ccp4bb] Solvent content of membrane protein crystals
Dear colleagues, in estimating the solvent content of membrane protein crystals it would only seem reasonable that micelle size should also be taken into account. Depending on the aggregation number and MW of a given detergent, the concentation of detergent used, and the buffer conditions, one may have micelles on the order of 15-25 kDa or even 35-50 kDa for detergents with alkyl chains of more than 10 carbons. However, when I took a look in a handful of papers reporting Matthews' numbers for membrane protein crystals, it became apparent that only the protein MW is used in such estimates. I am beginning to wonder if one should even bother reporting a Matthews number for a membrane protein crystal given the uncertainties surrounding size and role of micelles in crystal packing. Any thoughts on this? best wishes Savvas
Re: [ccp4bb] Solvent content of membrane protein crystals
I would use a very general definition for solvent, including disordered detergent and lipids. As you know in many cases ordered detergents and lipids have been modeled in the coordinates, so they are part of the model not the solvent. In some cases I think waters should be included in the model not solvent- say for structural waters buried in the protein at least. Ed Savvas Savvides wrote: Dear colleagues, in estimating the solvent content of membrane protein crystals it would only seem reasonable that micelle size should also be taken into account. Depending on the aggregation number and MW of a given detergent, the concentation of detergent used, and the buffer conditions, one may have micelles on the order of 15-25 kDa or even 35-50 kDa for detergents with alkyl chains of more than 10 carbons. However, when I took a look in a handful of papers reporting Matthews' numbers for membrane protein crystals, it became apparent that only the protein MW is used in such estimates. I am beginning to wonder if one should even bother reporting a Matthews number for a membrane protein crystal given the uncertainties surrounding size and role of micelles in crystal packing. Any thoughts on this? best wishes Savvas
