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
