[RASMB] value of analysis

[bchter at ksu.edu]

My question has to do with a relatively rapid semiemperical calculation
that seemed to give about the right result.  I want to know whether the
approach has any merit (e.g. describe result, footnote method) and whether
it has some theoretical basis or obvious defect. I have spelled out micro
since at least in my e-mail Greek letters do not work.

We are about to submit a paper on a 50 kDa protein that forms a well
behaved reversible dimer.  We have characterized this both by
sedimentation velocity and equilibrium (and the tighter binding to another
small protein shifting it to apparently all monomer).  SEDFIT gives a nice
fit to velocity profiles with a fairly narrow range of estimates of
equilibrium dissociation constants.  The average was average 8.6 + or -
1.3 micromolar for data at four protein concentrations (range 3.8 to 38
micromolar).  The change in RMSD with variation in Kd suggested less
accuracy for any one concentration but the average of appKd values at the
RMSD minima gave this Kd and error range.  The equilibrium data involved
four concentrations equilibrated at 3 speeds. The global fit of the
equilibrium data (using Beckman software) gave a Kd of 5.5 micromolar
(error range 4.7 to 6.0 micromolar).  We conclude that the Kd is probably
in the range of 4.7 to 10 micromolar.

Interestingly (at least to me) my initial estimate, made from the
sedimentation velocity data (data collected and above analysis by Yasuaki
Hiromasa), was within this range. I used the average dc/dt profiles
because I rationalized that the peak centers would be more sensitive to
dimer than g(s*) peaks. My analysis assumed that in the concentration
range where the peak center was between 30% and 70% of the maximal change
(estimated by extrapolation for all monomer and all dimer)  that the level
of dimer would be directly proportional to the proportion of change in
peak center position to maximal change.  The average apparent Kd was 7.9 +
or - 1.7 micromolar from this analysis of four sedimentation velocity runs
in the concentration from 1.9 to 19 micromolar.

Sorry so wordy.  I appreciate your comments.

Tom

Thomas E. Roche
University Distinguished Professor
Department of Biochemistry
Willard Hall
Kansas State University
Manhattan, KS 66506
bchter at ksu.edu
785-532-6116