Re: [ccp4bb] monomer/dimer protein
Hi Todd, Do you mean reversible or irreversible dimers? If the binding is reversible, then once crystal growth starts, Le Chatelier's principle should take over, pull the equilibrium toward monomer, and you should see minimal impact of dimer on your monomer crystals. The same would be true in reverse for proteins that pack as dimers or higher-order oligos in the crystal. If the dimer formation is irreversible (ie. disulfide-mediated, domain-swapped, ), they can be considered a chemically distinct entity and could indeed disrupt crystal packing. The most thorough treatment of the subject I know of is Chapter 8 of McPherson, although I don't think it addresses dimers specifically, just nonspecific aggregates. Perhaps still a place to start? Shane Caldwell McGill University On Wed, Aug 13, 2014 at 5:40 PM, Todd Jason Green tgr...@uab.edu wrote: Hello All- I am interested in monomer/dimer contamination when building a crystal lattice, ie. if you are building a crystal lattice with a monomeric species of protein, incorporation of dimers may yield lattice or surface defects. This species may be considered a macromolecular contaminant. I have read a few papers on this subject, a couple are listed here: I. Yoshizaki et al. / Journal of Crystal Growth 290 (2006) 185–191 Caylor CL1, Dobrianov I, Lemay SG, Kimmer C, Kriminski S, Finkelstein KD, Zipfel W, Webb WW, Thomas BR, Chernov AA, Thorne RE. Proteins. 1999 Aug 15;36(3):270-81. In each of the studies that I have read, lysozyme is the model protein for these studies. I have not seen studies thus far that have been done with other proteins. Can anyone point me toward other studies, specifically non-lysozyme studies, where incorporation of two different oligomerization states has been shown to yield crystals with higher level of defects? Microscopy studies of such would be great too. Thanks in advance- Todd
Re: [ccp4bb] monomer-dimer
Dear all, If I may add that I find the statement First, remember that gel filtration elution volumes are independent of conditions like flow rate and protein concentration (unless there are nonspecific interactions at high concentration), but like I described before temp is a factor. a bit misleading. While concentration will not change where the monomer or the dimer appears in the elution volume, concentration will affect the monomer-dimer equilibrium during your gel-filtration run. Thus, I would say that concentration is a factor. If your dimer has a kD of ~100uM, and you inject it at a concentration of ~100uM, after getting diluted during gel-filtration (about ten-fold) it will appear 90% as a monomer ... The results of any analytical technique to determine stoichiometry are concentration dependent, and concentration is actually the major variable that needs to be considered to define the oligomerization state (in AUC this can be done nicely). And do not forget that the in-vitro oligomerization state does not necessarily imply the same for in vivo, so please do make mutants to prove it before submitting the paper ... A. On Aug 10, 2010, at 1:38, Bostjan Kobe wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/ 10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is
Re: [ccp4bb] monomer-dimer
Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 11:45 AM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 19:30, chern wrote: Thank you for reply. 1.It will be nice to have mass-spec method for non-covalent complexes. Carol Robinson is doing these
Re: [ccp4bb] monomer-dimer
Hi ccp4bb Could you please send me some references with the sedimentation equilibrium calculations of Kd, monomer/dimer ratio etc. Maia Maia Cherney wrote: Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 11:45 AM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 19:30, chern wrote: Thank you for reply.
Re: [ccp4bb] monomer-dimer
Hi Maia, this review and website might be a good place to start: http://analyticalultracentrifugation.com/images/AUCinProteinScience.pdf http://analyticalultracentrifugation.com/default.htm 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 Hi ccp4bb Could you please send me some references with the sedimentation equilibrium calculations of Kd, monomer/dimer ratio etc. Maia Maia Cherney wrote: Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant.
Re: [ccp4bb] monomer-dimer
I agree completely with Anastassis that the equilibrium will be effected by changing the concentration of the sample during gel filtration, however I wanted to point out that the elution volumes of the two species are independent of their populations. I apologize if I was misleading. Mike - Original Message - From: Anastassis Perrakis a.perra...@nki.nl To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, August 11, 2010 2:10:16 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] monomer-dimer Dear all, If I may add that I find the statement First, remember that gel filtration elution volumes are independent of conditions like flow rate and protein concentration (unless there are nonspecific interactions at high concentration), but like I described before temp is a factor. a bit misleading. While concentration will not change where the monomer or the dimer appears in the elution volume, concentration will affect the monomer-dimer equilibrium during your gel-filtration run. Thus, I would say that concentration is a factor. If your dimer has a kD of ~100uM, and you inject it at a concentration of ~100uM, after getting diluted during gel-filtration (about ten-fold) it will appear 90% as a monomer ... The results of any analytical technique to determine stoichiometry are concentration dependent, and concentration is actually the major variable that needs to be considered to define the oligomerization state (in AUC this can be done nicely). And do not forget that the in-vitro oligomerization state does not necessarily imply the same for in vivo, so please do make mutants to prove it before submitting the paper ... A. On Aug 10, 2010, at 1:38, Bostjan Kobe wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20
Re: [ccp4bb] monomer-dimer
Hello Intekhab, Your results do not seem surprising at all. It is not uncommon for molecular interactions such as dimerization to be more stable at lower temperatures, and this is exactly why you are seeing the shift to higher elution volumes at lower tempratures. At lower temperatures, both the monomer and dimer are likely to be more compact in solution due to less thermal fluctuations in the overall structures. Remember that protein structures are always in motion, and lowering the temperature restricts these motions, and therefor lowers the effective radius of the molecule in solution as it moves through the column. And of course smaller molecules elute at higher volumes, so this probably explains what you see in the cold room. As for some of the other concerns you have with your gel filtration experiments, I can offer the following suggestions. First, remember that gel filtration elution volumes are independent of conditions like flow rate and protein concentration (unless there are nonspecific interactions at high concentration), but like I described before temp is a factor. That being said, often analytical gel filtration experiments are more informative at moderate concentrations instead of high concentrations, because this will favor the formation of relevant oligomers, instead of oligomers and aggregates that form only at high concentrations and aren't really biological. When you do your incubation experiments, try using lower protein concentrations or shorter incubation times. This might prevent the formation of precipitates and will give you more biologically relevant information - after all, most proteins are not available in the cell at very high concentrations, so if your dimer is biological, the kd is likely pretty low. Also you could try another experiment like a pulldown with tagged/untagged constructs, or SPR. These experiments (SPR particularly) would also tell you if the kd is reasonable for a biologically relevant interaction. One more thought is to be sure your protein is not degrading at high temperatures, which may be the reason your 37 degree incubation results in increased elution volume. Mass spec could help you here. Finally, to determine the biological relevance of your dimer you should do an analysis of the dimer interface seen in the crystal structure. I believe that for the average biological oligomer, the oligomerization interface buries approx 1200-2000A2 of surface area, whereas the average crystal contact buries approx 400-800A2. Some older work related to these analyses has been published by Joel Janin and Janet Thornton. Also, some webservers like PISA attempt to predict the relevant oligomerization states of proteins in the PDB based on interfaces seen in the crystal structures. You might look there for a good method. Good Luck, Mike Thompson - Original Message - From: intekhab alam faisal...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, August 9, 2010 4:37:45 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] monomer-dimer Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] monomer-dimer
To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] monomer-dimer
Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland.
Re: [ccp4bb] monomer-dimer
Dear That was a quite enlightening discussion!! I am grateful to you guys for your time!! I will definitily try some of these to get a clear answer. Regards Intekhab alam On Tue, Aug 10, 2010 at 8:38 AM, Bostjan Kobe b.k...@uq.edu.au wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail
Re: [ccp4bb] monomer-dimer
Small angle x-ray solution scattering (SAXS) can also give you molecular weight, though not quite as accurately as the best static light scattering. While SAXS is preferably done on monodisperse systems extrapolated to infinite dilution, cases in which the monomer and dimer are in equilibrium under various conditions can be treated ... especially if you already know the monomer and possible dimer structures. These are relatively easy experiments at a synchrotron source, but lab source SAXS facilities also can be found. Richard Gillilan MacCHESS On Aug 9, 2010, at 7:38 PM, Bostjan Kobe wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au
