[RASMB] viscosity issue in sedimentation velocity measurements of complex formation

[Donald W. Pettigrew]

Hello, Rasmb

 

I have a question about viscosity corrections for s20,w due to varying
concentrations of protein.

 

We are using sedimentation velocity to determine the binding constant for
association of an 18.1 kDa protein with a 224kDa protein.  The concentration
of the larger protein is maintained constant at 0.3 mg/mL (about 5 uM
subunits for the tetrameric protein) and the concentration of the smaller
protein is varied from 0-1 mg/mL (about 50 uM).  The binding stoichiometry
is known to be 1 small protein per subunit of the larger protein (4 per
tetramer).  The dissociation constant is known from independent studies to
be about 5 uM.

 

We run sedimentation velocity experiments at 28,500 rpm, using absorbance
optics at 280 nm, standard aluminum double sector cells.  We have the great
fortune that the smaller protein is almost entirely transparent at 280 nm.

 

At this juncture, we analyze the data by using SVEDBERG to obtain a single
value for s20,w.  We see the s20,w increase from about 11S to about 12+S as
the concentration of the smaller protein is increased 0-1 mg/mL.  This
increase is about what we expect.  We used the same corrections for s20,w at
all concentrations of the smaller protein, although the solution viscosity
should increase with increasing concentrations of the smaller protein.
Thus, we are underestimating the s20,w at the higher concentrations of the
smaller protein.  However, I have no feel for the extent of the error.

 

We are sort of following the paper: Dam et al (2005) Biophys J 89:619-634
from Peter Schuck, although we have not yet gotten to the elegant analysis
part.  I am not sufficiently familiar with the computational methods that
are described in the paper to see whether a correction for viscosity is
there.

 

We can determine empirically the viscosity effect on sedimentation of the
larger protein by using mutants of it that we know do not bind the smaller
protein.  However, I wonder whether this is sufficient or are there better
ways to correct for the viscosity differences?  If so, what are they?  Does
anyone on the listserv know about this issue?

 

Thanks for your consideration of this lengthy email.

 

Dr. Donald W. Pettigrew

Department of Biochemistry & Biophysics

Texas A&M University

College Station, TX 77843-2128

 

phone: 979-845-9621

fax: 979-845-9274

 

"Nearly all  men can stand adversity, but if you want to test a man's
character, give him power"

A. Lincoln

 

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