[RASMB] 1)Diffusion coefficient and 2) ATP

[Jo Butler]

Dear Songpon,

As someone who has worked with nucleic acids, I feel able to reply to your 
second point.

ATP is a nucleotide triphosphate and its absorption maximum will be around 
260nm (not 280 as you suggest, although the tail extends out here). 
However, it will also absorb strongly at 230nm and, with 200:1 ATP to 
protein your whole experiment will be swamped by the absorbance of the free 
ATP.  The actual Mr depends on the exact salt, but for most likely forms it 
is ~500Da, so you cannot expect any significant redistribution of free ATP 
during sedimentation - and even if your protein binds say 2 ATP, you will 
still have 99% of the ATP unliganded.
A further complication is that NTPs are labile to hydrolysis, both by any 
contaminating enzymes and also simply by the water of your buffer.  This is 
why many experiments are conducted with the non-hydrolysable sulphur and 
methylene variants of NTPs, rather than the natural phosphates.  Thus as 
your experiment runs, you will have steadily less ATP and more ADP, or even 
AMP, present.  With the initial excess of ATP this may not matter for the 
binding, but you should be aware of this as the lower phosphate forms may 
compete with the triphosphate.

Overall, I am afraid that I have grave doubts about whether your 
sedimentation equilibrium experiment will be interpretable.

Sorry,

Jo

--On Monday, September 23, 2002 5:30 pm -0700 songpon <songpon at scripps.edu> 
wrote:

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> 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
>
>
>
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P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296