[RASMB] analysis of heterodimerization

[Glen Ramsay]

Greetings Satinder:

The AU-FDS fluorescence detector developed by UNH might help.  Excitation 
is based on a 488 nm laser, which excludes native fluorescence.  An 
experiment might look like this:  Label the B protein with Alexa.  "A" 
monomer and AA dimer will be invisible as they will not absorb 488 nm 
light.  B monomer will fluoresce, but will clearly distinguished from 
dimers.  BB dimer has been shown to not exist at these concentrations so it 
can be ignored.  AB dimer will fluoresce.  Viola!

Disclaimer: Aviv Biomedical is commercializing the AU-FDS.  Contact me if 
you wish more information.

Glen

At 11:18 PM 3/28/2005, you wrote:
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>
>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
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    Glen Ramsay, Ph.D.
    Chief Scientist
    Aviv Biomedical
    750 Vassar Avenue
    Lakewood, NJ 08701
    (732) 370-1300, Ext. 25
    (732) 370-1303, FAX
    glen at avivbiomedical.com
    www.avivbiomedical.com