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 <[log in to unmask]
>> <mailto:[log in to unmask]>> 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:[log in to unmask]>
>> *To:* chern <mailto:[log in to unmask]>
>> *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:[log in to unmask]>
>>> *To:* chern <mailto:[log in to unmask]>
>>> *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:[log in to unmask]>
>>>> *To:* [log in to unmask]
>>>> <mailto:[log in to unmask]>
>>>> *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" <[log in to unmask]
>>>>> <mailto:[log in to unmask]>>
>>>>> To: <[log in to unmask]
>>>>> <mailto:[log in to unmask]>>
>>>>> 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" <[log in to unmask]
>>>>> <mailto:[log in to unmask]>>
>>>>> >>> To: <[log in to unmask]
>>>>> <mailto:[log in to unmask]>>
>>>>> >>> 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
>>>>> >>>>
>>>>> >
>>>>
>>>> *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
>>>>
>>>>
>>>>
>>>>
>>>>
>>>
>>>
>>
>>
>
>
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