Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation
Dear Jerry, Just a few comments on your questions. We tried both SV and SE. SV showed an increasing Sw with decreasing concentrations (Sw varied from 6.9, 7.0, 7.2, 7.4 to 7.522). IN addition, the SV experiments showed a single boundary. However, the boundary was a little broader, between 6 and 8.5. SV peaks seem to overlay at lower concentrations with Sw ranging from 7.5 to 7.86. From SV experiments, the calculated Mw is ~125 KD with f/fo equal to 1.2. The monomer of my protein is ~20KD. I am not sure whether 7.86 S would be much faster than a tetramer (~80KD) could possibly sediment. Your data seems to point towards non-ideal behaviour of your protein, more precisely towards repulsive protein-protein interactions. Keep in mind that Mw is an apparent mass that depends on interactions with the solvent and is protein-concentration dependant (in addition to the issue of oligomers). For an example, you could check : Solovyova, A. et al. (2001). Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents. Biophys. J. 81:1868-1880. http://dx.doi.org/10.1016/S0006-3495(01)75838-9 I would suggest you to post your questions to the RASMB email list info that is centred on AUC and with very helpful people. Best regards, Lionel
Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation
Dear Kushol Jerry, I have to take exception to Kushol's contention about SV. As long as the buffer and protein parameters are correct and the sample is well behaved (i.e. not undergoing dynamic rearrangement on the time scale of the SV experiment, not aggregated, homogeneous), one can derive very accurate molar masses from SV, and it is far superior in this respect than SEC. However, as you aptly point out, the problem may lie in the sample's behavior. If the protein is populating multiple oligomeric states, or if it is undergoing a fast interconversion of such states, or if it is not pure, spurious masses may be calculated. If such pathologies are observed, it's not clear to me that a sedimentation equilibrium experiment would help. For a complicated interaction model, SE data (and AUC data in general) can be difficult to analyze, and some model assumptions are almost always present. So, the questions are: 1. Did Jerry run SV or SE? 2. If the former, then did he see a nice, single boundary, or a big smear? 3. If the latter, how were the data analyzed to come to his conclusion? Hopefully he can comment on these aspects. Cheerio, Chad From: Kushol Gupta kushol.gu...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, May 27, 2011 9:51:04 AM Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Hi Jerry – By AUC, do you mean sedimentation velocity (SV)? Both gel filtration and SV are not terribly great ways to determine precise molecular mass, especially if the macromolecule of interest is anisotropic in shape. In your SV values, do you see a large f/fo, or a broad distribution? Can you run a sedimentation equilibrium experiment? If you run HYDROPRO on your prospective oligomer structure, do you arrive at theoretical S and Rs values that jive with your solution data? A nice crosscheck you could do with the data in hand (if your measurements from both approaches were performed in the same buffer at the same temperature) is calculate the mass using the Siegel and Monty equation (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346–362), where the mass of the particle is calculated from Rs (from gel filtration) and the S(t,b) value from sedimentation velocity. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From:CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state. Could anyone give some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully
Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation
Dear Kushol, Chad and other folks; Thanks a lot for the comments. I am sorry for keeping silent because we are still repeating the SE experiment. However, here are some preliminary data. Did Jerry run SV or SE? 2. If the former, then did he see a nice, single boundary, or a big smear? We tried both SV and SE. SV showed an increasing Sw with decreasing concentrations (Sw varied from 6.9, 7.0, 7.2, 7.4 to 7.522). IN addition, the SV experiments showed a single boundary. However, the boundary was a little broader, between 6 and 8.5. SV peaks seem to overlay at lower concentrations with Sw ranging from 7.5 to 7.86. From SV experiments, the calculated Mw is ~125 KD with f/fo equal to 1.2. The monomer of my protein is ~20KD. I am not sure whether 7.86 S would be much faster than a tetramer (~80KD) could possibly sediment. In the meanwhile, light scattering on my sample showed a radius equal to a tetramer. 3. If the latter, how were the data analyzed to come to his conclusion? At present, we also want to measure the Vpar in order to better fit the SE data. Any suggestions here? The problem is that we only observed tetramers in the crystals which were grown from PEG, which probably would keep the protein’s native oligomerization state. How could we interpret the discrepancy? Thanks again and welcome more comments and suggestions. Jerry Date: Thu, 2 Jun 2011 11:37:57 -0700 From: cabrautc...@yahoo.com Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation To: CCP4BB@JISCMAIL.AC.UK Dear Kushol Jerry, I have to take exception to Kushol's contention about SV. As long as the buffer and protein parameters are correct and the sample is well behaved (i.e. not undergoing dynamic rearrangement on the time scale of the SV experiment, not aggregated, homogeneous), one can derive very accurate molar masses from SV, and it is far superior in this respect than SEC. However, as you aptly point out, the problem may lie in the sample's behavior. If the protein is populating multiple oligomeric states, or if it is undergoing a fast interconversion of such states, or if it is not pure, spurious masses may be calculated. If such pathologies are observed, it's not clear to me that a sedimentation equilibrium experiment would help. For a complicated interaction model, SE data (and AUC data in general) can be difficult to analyze, and some model assumptions are almost always present. So, the questions are: 1. Did Jerry run SV or SE? 2. If the former, then did he see a nice, single boundary, or a big smear? 3. If the latter, how were the data analyzed to come to his conclusion? Hopefully he can comment on these aspects. Cheerio, Chad From: Kushol Gupta kushol.gu...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, May 27, 2011 9:51:04 AM Subject: Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Hi Jerry – By AUC, do you mean sedimentation velocity (SV)? Both gel filtration and SV are not terribly great ways to determine precise molecular mass, especially if the macromolecule of interest is anisotropic in shape. In your SV values, do you see a large f/fo, or a broad distribution? Can you run a sedimentation equilibrium experiment? If you run HYDROPRO on your prospective oligomer structure, do you arrive at theoretical S and Rs values that jive with your solution data? A nice crosscheck you could do with the data in hand (if your measurements from both approaches were performed in the same buffer at the same temperature) is calculate the mass using the Siegel and Monty equation (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346–362), where the mass of the particle is calculated from Rs (from gel filtration) and the S(t,b) value from sedimentation velocity. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state
[ccp4bb] larger molecular weight shown by analytic ultracentrifugation
Dear ALL; I am sorry for this off-topic question about analytic ultracentrifugation (AUC). We recently solved one structure from crystals grown out of PEG4000 plus buffer. Since the crystal was grown from PEG, we think the protein would maintain its native oligomerization state as in the solution. Indeed, the crystal packing clearly shows a tetramer of this protein. However, both the gel-filtration and AUC showed larger molecular weight, roughly around 6-mer or 7-mer. IN the crystal lattice, we could not find any 6-mer or 7-mer state. Could anyone give some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully
