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>
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
>>>>
>
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
