[RASMB] Sw Isothem Fitting in SEPHAT using Fluorescence Data

[David Chiovitti]

Hi everyone,

I am studying a tRNA-protein binding interaction using the AVIV AU-FDS
system.  I am using a fixed amount of fluorescently labelled tRNA and
titrating in the protein.  From the c(s) plots I have determined that it is
a slow exchange between the protein and the tRNA.  The tRNA give a peak at
approximately 3.5 S.  As I titrate in the protein the 3.5 S peak decreases
and a new peak at 4.9 S appears.  This is consistent with one tRNA being
bound by one protein.  For each titration I have used a weighted vbar value
based on the amount of protein and tRNA.  I have been using the vbar value
of 0.530 for the tRNA.

For each of the titrations I have integrated the peaks to determine a
weighted signal value and put them into a text file according to the
formatting listed on the SEPHAT website.  I open the file up with no issue
in SEDPHAT and select the hetero-assocation model.  I enter the molecular
weight and S values of each of the components and float the logKa value,
which I set to 5.  When I run and fit the data I get a curve that doesn't
even come close to fitting the data points.  I'm really at a loss to what
I'm doing wrong.

Here is the isothem data:
0.82 0.1 3.6
2.74 0.1 3.48
7.4  0.1 3.50
22.7 0.1 4.0
66.6 0.1 4.94
200 0.1 4.90

The first value being the concentration of the protein, the second the
concentration of the tRNA, and the third is the Sw value.

Other than SEDPHAT is there another program I could use to fit this
Isotherm?

Thanks,
David Chiovitti

Samuel Lunefeld Research Institute
Mount Sinai Hospital Toronto
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