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]>
>> To: <[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
>>>