[RASMB] analysis of heterodimerization

[ss2337 at columbia.edu]

Hello,

I have a question about analyzing a heterodimeric interaction. We
have collected data using 2 techniques, SEC/LS (size exclusion
chromatography coupled to light scattering) and sedimentation
equilibrium.

We have 2 proteins, A & B.
The molecular weight of A is 33.3 kDa, and the molecular weight of B
is 31.8 kDa. Another important property is that they both have
approximately the same extinction coefficient at 280 nm, i.e., they
have about the same numebr of tryptophans & tyrosines.

When A is analyzed by SEC/LS, the peak (dimer) that comes off the
column and is analyzed by light scattering is a mixture of monomer
and dimer. WHen B is subjected to the same analysis, it is only
monomer. However, when A and B are mixed in an equimolar ratio, the
"B" peak disappears and shifts into the dimer peak of A.
Our interpretation was that A and B are heterodimerizing.


To acquire some quantitative data on the interaction, we went to
sedimentation equilibrium and did the following analysis using
6-sector cells at 280 nm at 4C:

1. Protein A alone at 3 different concentrations (0.2, 0.4, 0.8
mg/ml) and 3 speeds (13k, 18k, 25k rpm). The data were analyzed
using WinNONLIN, several models were tried, and the best model was
a monomer-dimer equilibrium with an SRV of 4.9x10-3 and very nice,
i.e, random, residual plots. The Kd was calculated to be 22.4 uM.

2. Protein B alone at the same concentrations and speeds. THe data
were analyzed again with WinNONLIN and could best be fit to a
single species monomer. I tried other models, like single species
monomer, mon-dim, etc., but the residuals were all VERY skewed. The
SRV with the single species monomer was 4.2x10-3 and the residual
plots looked very nice. So, I am pretty sure this is the best
model.

3. Proteins A & B in an equimolar ratio using a TOTAL protein
concentration of 0.2, 0.4, 0.8 mg/ml and the same 3 speeds. We
analyzed the data using WinNONLIN using the same type of analysis
described above and came up with a Kd of 30.3 uM.

Now here are the questions:
1.In the mixture, both homoassociation (A+A --> AA) and
heteroassociation (A+B --> AB) are taking place, so what does the
calculated Kd of 30.3 uM represent? I assume no BB homoassociation
is taking place because it doesn't occur alone (see point 2 above).
We know what the Kd of AA homoassociation is. Can we calculate a
heterdimer Kd from these data??

2. Does the Kd of 30.3 uM represent anything at all or is it just
garbage because I don't think WinNONLIN in equipped to handle
heteroassociations.

3. What would be the best way of calculating the Kd of the
heterodimer in light of the data we already have, i.e., can we use
the data we already have (SEC/LS & AUC) or do we need to do more
experiments? If we can use the data we already have, how would you
calculate the heterodimer Kd? If we need to do more experiments,
should they be equilibrium or velocity? If we do SE, what program
should I use? If we use SV, what program should I use and how do
you calculate a Kd from velocity data?

Thank you all in advance for your help.

Sincerely yours,
Satinder K. Singh