[RASMB] combining DLS and sedimentation equilibrium to getmolecular shape

[John Philo]

Dave,

I want to address a couple of additional technical points, but first I
should say I agree with Peter that DLS may over-estimate the true diffusion
coefficient, especially if you are just using the z-average radius value
from the cumulants method.

I am surprised you would say you didn't have enough material for a velocity
experiment. One can easily get the sedimentation coefficient with more than
sufficient accuracy using only 2 micrograms of protein (or less) and a
conventional full 1.2 cm column if you scan at 230 nm. You don't need to be
able to visibly see a boundary over the noise, and pretty much all of the
software packages can handle it. For example, I've done this with as little
as 0.5 microgram total protein using DCDT+, and still had sufficient
signal/noise to show the sample was heterogeneous.

Second, regarding how to calculate f/f0 from your data, in my opinion for
best accuracy you should use the sequence mass (or the appropriate integer
multiple if the native state is an oligomer) in the calculations rather than
the experimental value from SE. 

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of David Lee
Sent: Wednesday, March 15, 2006 7:22 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] combining DLS and sedimentation equilibrium to getmolecular
shape


Dear colleagues,
I am writing to enquire about the validity and pitfalls of modeling  
hydrodynamic shape by combining sedimentation equilibrium data with  
dynamic light scattering data.  I didn't have enough protein for  
sedimentation velocity experiments so after performing sedimentation  
equilibrium experiments, whcih showed the complex to behave as a  
single ideal species, I measured the hydrodynamic radius by dynamic  
light scattering, then calculated the frictional coefficient f.  I  
combined this number with the molecular weight obtained from the  
equilibrium experiment to  calculate s* which I entered along with  
the other data into SEDNTERP  to model the dimensions of the protein  
as a prolate ellipse.  Is this a valid way of  doing hydrodynamic  
modeling?  I'm worried that I'm overlooking something.  Thanks in  
advance for your input on this matter.

Sincerely,
Dave

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