[RASMB] Possible for a complex to break apart from force of sedimentation?

Laue, Thomas Tom.Laue at unh.edu
Tue Dec 15 06:19:19 PST 2015


Hi-
AUC doesn’t lie… If the complexes are near or in the void volume, SEC won’t provide much resolution. Likewise, DLS is fitting the correlation function the best it can, and it will fit it with a single decay if that suffices to describe the data.
There is insufficient force across a complex for it to be torn apart by the gravitational field. The force available will be the gradient of the gravitational potential energy, with the diameter of the complex being the distance, which is very short. On the other hand, if you suspect pressure effects in the AUC, you have some choices for diagnosis.

1.       The pressure effect depends on the number of units affected (the stoichiometry of the complex), which is moderately high for a MDa complex of 60 KDa subunits. Pressure usually leads to dissociation of complexes. The pressure is non-uniform across the cell, and depends on the rotor speed. You should get horrible fits to the data since dissociation will be greater towards the base of the cell. Also, the extent of dissociation will be rotor speed dependent (by the square of the rotor speed). See if the distribution shifts towards larger complexes at lower rotor speeds. If the distributions are insensitive to rotor speed, you have a good indication that the distribution of sizes that is pressure independent.

2.       Layer a bit of mineral oil over the sample. This will add static pressure to the sample. If the distribution is pressure dependent, you will see greater dissociation when you analyze the data.
Best wishes,
Tom

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Toth IV, Ronald T
Sent: Monday, December 14, 2015 5:15 PM
To: rasmb at rasmb.org
Cc: Kumru, Ozan S
Subject: [RASMB] Possible for a complex to break apart from force of sedimentation?


Hi all! We’re looking at a ~60 kDa protein that forms a ~ 1 MDa oligomeric disulfide linked complex. By SEC and DLS the sample is very monodisperse at about what we would expect the MDa complex to look like. However by AUC we see a very heterogeneous distribution between 5s and 30s. I was wondering if anyone else had observed a system where a complex had broken apart from the force of sedimentation?



Thanks in advance!   ​




- Ronald Toth

Research Associate
Macromolecule and Vaccine Stabilization Center
University of Kansas
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