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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