[RASMB] 1)Diffusion coefficient and 2) ATP

[songpon]

Dear all:

I have two separate questions for two separate experiments.   I will 
appreciate any input on either or both questions.

1) I am studying a rod shape protein.  Upon mutation, we suspect that the 
protein will tighten up and we expect that the hydrodynamic property would 
change.  I ran sed velocity experiment and fit the data by DCDT.  The result 
showed that the S value is higher for the mutant (7.2 vs 6.8).  On the other 
hand, the diffusion coefficients are very different (27 for mutant and 9 for 
wild-type).  My questions for this experiment are:
     a) How can I interpret the difference in diffusion coefficients?
     b) What is the physical meaning of diffusion coefficient for AU 
experiment?

2) I am studying membrane protein oligomerization upon addition of ATP.  I did 
it by sed equilibrium.  The amount of ATP to protein is 200:1 which is 
standard for this family of protein.  I had no probelm at all running the 
protein alone in presence of detergent (0.1% LDAO).  However, when I run the 
sample that contain ATP, I observed that wavelength scan only show absorbance 
for ATP (at 280 nm), but not for peptide bond (at 230 nm).  In addition, I 
observed that the signal become messy and un-analyzable as the equilibrium run 
progressed.  What I saw were:
     i) Baseline increased from 0.3 AU to 1.0 AU
     ii) No traditional boundary (exponential-like shape) was observed.  The 
boundary looked more like a broad parabolic curve.
     My questions are:
     a) Is ATP interference the explanation for the observation?
     b) If this is the case, how can I prevent it?

I really appreciate your time reading this long email.  I appreciate all the 
response you gave me for my previous questions as well.  Thank you very much.

Sincerely
Songpon Deechongkit
The Scripps Research Institute
Kelly Lab
La Jolla, California