It's an interesting discussion. 1.Usually it's not possible to use mass-spec for non-covalent complexes.
2. Most methods depend on macromolecule shape and concentration. 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? 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. 5. There are papers on capillary electrophoresis methods where they study exactly these effects (time of equilibrium vs time of separation). Maia ----- Original Message ----- From: "aidong" <a...@xmu.edu.cn> To: <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> >>> To: <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/multicoil >>>> ) 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 >>>> >
