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. >>> 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 routinely ... so does >>> Albert Heck >>> >>>> 2. 10nM solution is very dilute to >>>> catch most >>>> complexes. So methods that work only for diluted >>>> solutions are not reliable to determine the >>>> multimeric state. What about > 100 mkM solutions >>>> for Kd`10^(-4)M? >>> >>> But its all matter of kD!! If my kD is 1nM, I will >>> see it at 10 nM. If you have 1 mM kD, then you need >>> 100mM sample! >>> Most methods you can measure what you want, its a >>> sample preparation issue. >>> >>>> 3. For study of multimers in AUC. >>>> If you have two >>>> separate bands, one of a monomer and one of a dimer, >>>> there will be continuous association in the monomer >>>> band and continuous dissociation in the dimer band >>>> even while they are separating, so there will be >>>> continuous migration between two bands and smearing. >>>> That's what I meant. AUC never worked for >>>> determination of multimeric state for me. I got many >>>> times uninterpretable results. I know that it works >>>> sometimes, most likely when teq>>tsep (who knows >>>> what t(eq) is for different proteins), even with >>>> some broadening of the bands. >>> >>> I really do not agree with that. Are you talking >>> about equlibrium or sedimentation runs? I agree it >>> does not always work well, but that is mostly >>> misbehaving samples over large times in eg the wrong >>> temperature, rather than anything else. A complex >>> with kOFF of about a minute or two can be measured >>> over weeks in AUC, the one is unrelated to the other. >>> btw, I still dont get what you mean t(eq). The only >>> definition of the time you need to reach equilibrium >>> in a system is infinity. >>> >>> A. >>> >>> >>> >>>> >>>> Maia >>>> >>>> ----- Original Message ----- >>>> *From:* Anastassis Perrakis >>>> <mailto:a.perra...@nki.nl> >>>> *To:* CCP4BB@JISCMAIL.AC.UK >>>> <mailto:CCP4BB@JISCMAIL.AC.UK> >>>> *Sent:* Monday, July 05, 2010 9:36 AM >>>> *Subject:* Re: [ccp4bb] monomeric coiled >>>> coil--updated >>>> >>>> >>>> On Jul 4, 2010, at 20:35, chern wrote: >>>> >>>>> It's an interesting discussion. >>>>> 1.Usually it's not >>>>> possible to use mass-spec >>>>> for non-covalent complexes. >>>> >>>> Native mass spec is well developed. I would hope >>>> to give you my favorite reference, >>>> but its for now pending, courtesy of some >>>> reviewers in Mol. Cell. ;-) >>>> Seriously now, native mass spec is a very nice >>>> option and the work on non-covalent complexes >>>> by Carol Robinson and others is good proof for >>>> that. >>>> >>>>> 2. Most methods depend on macromolecule shape >>>>> and concentration. >>>> >>>> Yes, but we need to be specific, for >>>> example, MALLS is shape independent, unlike SEC >>>> which is size dependent. >>>> I would add that most methods depend on having a >>>> good estimate of concentration. The result you >>>> get for your system, not the method itself, >>>> is concentration dependent: eg MALLS is >>>> perfectly fine for a 10nM complex, but utterly >>>> useless for a 10mM "complex". >>>> AUC has the advantage to work across a wide >>>> range of expected kD's although some tuning is >>>> needed. >>>> >>>>> 3. SAXS method looks limited to me. It uses >>>>> diluted monodisperse solutions. That excludes >>>>> complexes that can associate/dissociate. How >>>>> can you calculate Kd from it? >>>> >>>> Yes, although its not the best way. Since you do >>>> measure at different concentrations, if you are >>>> above-below the kD so in the low end >>>> you have monomer and in the higher end dimer, >>>> you can estimate well the kD. >>>> >>>>> 4. All methods for determination of multimeric >>>>> state using separation technique depend on >>>>> three different cases: time of equilibrium >>>>> (teq)>> time of separation (tsep), or teq << >>>>> tsep or teq ~ tsep. Even without equilibrium, >>>>> you cannot have only one component. For a >>>>> successful separation, you would want teq >> >>>>> tsep, which is less likely in AUC method. >>>> >>>> I guess there is a confusion of two different >>>> issues: >>>> >>>> 1. Separation of multimers: To separate two >>>> things, indeed as you put it, you want >>>> teq>>tsep, and I would add that you want that >>>> for a the concentration(s) that your separation >>>> method works. In general, to separate two >>>> things, you want to be at a concentration where >>>> one state is clearly preferred over the other. >>>> >>>> 2. Study of multimers: In AUC for example you >>>> study the presence of multimers as a function of >>>> time, so you can observe all kinds of multimers >>>> and from their relative abundance you can study >>>> the association characteristics. >>>> >>>> >>>>> 5. There are papers on capillary >>>>> electrophoresis methods where they study >>>>> exactly these effects (time of equilibrium vs >>>>> time of separation). >>>> >>>> A. >>>> >>>> >>>>> Maia >>>>> >>>>> ----- Original Message ----- >>>>> From: "aidong" <a...@xmu.edu.cn >>>>> <mailto:a...@xmu.edu.cn>> >>>>> To: <CCP4BB@JISCMAIL.AC.UK >>>>> <mailto:CCP4BB@JISCMAIL.AC.UK>> >>>>> Sent: Sunday, July 04, 2010 3:01 AM >>>>> Subject: Re: [ccp4bb] monomeric coiled >>>>> coil--updated >>>>> >>>>> > In light of several wonderful responses,I >>>>> would like to provide an >>>>> > update for this question: >>>>> > > 1. I would agree that >>>>> SEC might not be able to >>>>> identify monomer vs > >>>>> multimer forms for this likely rod-shaped protein. >>>>> > > 2. It is extremely low >>>>> kd for dimer. AUC and >>>>> SAXS experiments have > >>>>> measured its kd at ~0.1 mM. >>>>> > > 3. MALS might not be >>>>> able to pick up dimer >>>>> form since it might be only >>>>> > a few percent when the concentration is low. >>>>> We might overcome > >>>>> concentration effect by direct injection to >>>>> dawn heleos and refraction >>>>> > index. >>>>> > > 4. Mass spec has found >>>>> both monomer and dimer >>>>> forms although the > >>>>> abundance of each one is not known. >>>>> > > 5. Intramolecular >>>>> coiled coil is quite >>>>> possible since intermolecular >>>>> > dimer is unstable. We hope our structure might >>>>> provide an answer. >>>>> > > Many thanks for your >>>>> time and ideas >>>>> > > Cheers >>>>> > > Aidong >>>>> > > > >>>>> On Jul 4, 2010, at 1:09 AM, Anastassis >>>>> Perrakis wrote: >>>>> > >> A few thoughts on >>>>> these, since I do not fully >>>>> agree. >>>>> >> >>>>> >> 1. Detection by light scattering is a method >>>>> that can be used either >>>>> >> without separation, or while separating. >>>>> >> If you have a scattering detector, you can >>>>> stick in a cuvette, or >> >>>>> stick it to the end of a column, your choice. >>>>> >> >>>>> >> 2. Sec is not a good method to show if >>>>> especially a coiled coil is >>>>> >> monomer-multimer. A long coil, will >>>>> >> have a hydrodynamic radius bigger than its >>>>> MW, thus any prediction >>>>> >> based on SEC will be misleading, >>>>> >> especially for this class of proteins. >>>>> >> >>>>> >> 3. In AUC (although I am not an expert at it >>>>> at all) I cant see the >> >>>>> connection between the disassociation time >>>>> >> and the run time. In sedimentation or >>>>> equilibrium runs, depending on >>>>> >> what you want to see, I think you can look >>>>> >> at monomer-multimer equilibrium over a wide >>>>> range of kD and >> >>>>> combinations of k(on) and k(off). >>>>> >> >>>>> >> 4. The physiological concentration is a bit >>>>> misleading. First, its >> >>>>> clear now that cells have microenvironments, >>>>> >> and 'physiological' concentrations are hard >>>>> to define. Also, in a >> >>>>> cell, I think (and I think others tend to agree) >>>>> >> that kD plays little role at the end. kD is a >>>>> combination of k(on) - >> >>>>> which is concentration dependent but in a cell >>>>> >> very likely diffusion limited - and of k(off) >>>>> which I think is what >> >>>>> matters most in the cell. >>>>> >> >>>>> >> Going to Aidong's question, I think that >>>>> MALLS was a good >> >>>>> experiment. The fact that these constructs do >>>>> no associate, >>>>> >> can mean that >>>>> >> >>>>> >> a. the prediction is wrong - likely with >>>>> these scores, but not >> >>>>> necessary >>>>> >> b. the kD in solution is indeed higher that >>>>> the concentration you >> >>>>> used for MALLS >>>>> >> c. The constructs are not well chosen for >>>>> some reason >>>>> >> >>>>> >> You could use AUC to detect kD as high as >>>>> ~100uM, depending on the >>>>> >> concentration of the start sample of course. >>>>> >> The next question will anyway be if that kD >>>>> has any sort of >> >>>>> physiological significance - which you cannot >>>>> tell by magnitude - >>>>> >> so you are back at the drawing board for >>>>> mutants. Three years later >>>>> >> the referees will still not believe it ... >>>>> sorry, now it gets >> >>>>> personal, >>>>> >> so I stop here. >>>>> >> >>>>> >> My two cents. >>>>> >> >>>>> >> A. >>>>> >> >>>>> >> >>>>> >> On 3 Jul 2010, at 18:10, chern wrote: >>>>> >> >>>>> >>> The multimeric state depends on a protein >>>>> concentration. You can >>>>> >>> get any >>>>> >>> multimer to dissociate if you dilute it to >>>>> low enough >>> >>>>> concentration. If >>>>> >>> your complex is a homodimer, then >>>>> Kdiss=[complex]/[monomer]^2. >>>>> >>> Let's say >>>>> >>> your Kdiss~10^(-3)M, and your protein >>>>> concentration is ~10^(-4)M, >>>>> >>> then >>>>> >>> >>>>> >>>>> [complex]=Kdiss/[monomer]^2=10^(-3)/10^(-4)^2=10^(-5), >>>>> that means, >>> the dimer >>>>> >>> concentration is approximately ~10 times >>>>> less then the monomer >>> >>>>> concentration >>>>> >>> at this particular protein concentration. >>>>> Let's say, the mol weight >>>>> >>> is 50 >>>>> >>> kDa, then at 5mg/ml you will have only about >>>>> ~10% of the dimer. Of >>> >>>>> course, >>>>> >>> if your Kdiss~10^(-4)M, then you will have >>>>> approximately similar >>>>> >>> concentrations of monomers and dimers at >>>>> 10^(-4). >>>>> >>> Because this is a dynamic equlibrium between >>>>> multimers and >>> >>>>> monomers, some >>>>> >>> methods are not good for the determination >>>>> of a multimeric state. >>>>> >>> Some >>>>> >>> reviewers demand to prove the multimeric >>>>> state by size-exclusion >>>>> >>> chromatography (SEC) or analytical >>>>> centrifugation. The analytical >>>>> >>> ultracentrifugation method will not work, as >>>>> the characteristic >>> >>>>> time of the >>>>> >>> dissociation/association is much lower than >>>>> the centrifugation time >>>>> >>> (`24 >>>>> >>> hours). The separated monomer will start >>>>> association and the >>> >>>>> separated dimer >>>>> >>> will start dissociation according to Kdiss >>>>> and the bands will be >>> >>>>> smeared. >>>>> >>> SEC is faster, like half an hour, it gives >>>>> you a better chance. The >>>>> >>> methods >>>>> >>> without separation are the best Like light >>>>> scattering), just make >>>>> >>> protein >>>>> >>> concentration high. Here comes the other >>>>> question. What is the >>> >>>>> physiological >>>>> >>> concentration. You want to be close to it. I >>>>> read some literature >>> >>>>> on this >>>>> >>> and it looks like it is between 10^-(4) to >>>>> 10^-(6) for majority of >>>>> >>> proteins. >>>>> >>> >>>>> >>> >>>>> >>> >>>>> >>> >>>>> >>> >>>>> >>> >>>>> >>> >>>>> >>> ----- Original Message ----- >>>>> >>> From: "aidong" <a...@xmu.edu.cn >>>>> <mailto:a...@xmu.edu.cn>> >>>>> >>> To: <CCP4BB@JISCMAIL.AC.UK >>>>> <mailto:CCP4BB@JISCMAIL.AC.UK>> >>>>> >>> Sent: Saturday, July 03, 2010 6:26 AM >>>>> >>> Subject: [ccp4bb] monomeric coiled coil >>>>> >>> >>>>> >>> >>>>> >>>> Sorry for this ccp4 unrelated question. >>>>> >>>> >>>>> >>>> We recently have a protein that a multicoil >>>>> program >>>>> >>>> >>>>> >>>>> (http://groups.csail.mit.edu/cb/multicoil/cgi-bin/multicoil.cgi/cgi-bin/mult icoil >>>>> >>>>> >>>> ) predicts to have very high probability >>>>> for dimer and trimer. >>>>> >>>> Their >>>>> >>>> scores are close to 0.4 and 0.6 for lengths >>>>> of more than 60 amino >>>>> >>>> acids. >>>>> >>>> However, two constructs that cover this >>>>> region have demonstrated >>>>> >>>> monomers >>>>> >>>> in solutions by Multiangle light >>>>> scattering?! For the same >>>>> >>>> question, we >>>>> >>>> could not get any response from this >>>>> program manager therefore we >>>>> >>>> turn to >>>>> >>>> ccp4 for help. We wonder whether some of >>>>> you might have similar >>>>> >>>> experience. Thank you in advance. >>>>> >>>> >>>>> >>>> Sincerely, >>>>> >>>> >>>>> >>>> Aidong >>>>> >>>> >>>>> > >>>> >>>> *P** **please don't print this e-mail unless you >>>> really need to* >>>> Anastassis (Tassos) Perrakis, Principal >>>> Investigator / Staff Member >>>> Department of Biochemistry (B8) >>>> Netherlands Cancer Institute, >>>> Dept. B8, 1066 CX Amsterdam, The Netherlands >>>> Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile >>>> / SMS: +31 6 28 597791 >>>> >>>> >>>> >>>> >>>> >>> >>> >> >> > >
