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" <[email protected]>
To: <[email protected]>
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
