From: Hilal A. Lashuel <Hilal@scripps.edu>
  To  : rasmb@bbri.harvard.edu
  Date: Wed, 08 Jul 1998 20:01:43 -0700

help with s values of membrane proteins in detergents

We would appreciate if anyone can help us address the situation outlined
below. 

We are currently characterizing an integral membrane protein by analytical
ultracentrifugation as well as other size determination 
methods (eg gel filtration) in detergent solutions (0.5% CHAPS).  We have
observed that deletion of the single predicted 
transmembrane domain (approx. 30 amino acids) drastically affects the
observed s values in sedimentation velocity.  The 
transmembrane domain containing protein exhibits an a heterogeneous range
of s values from 17-25 S, while the 
transmembrane deleted protein behaves as a single 11S species (both
proteins, however require the presence of detergents).  
These observations are confirmed over a 10 fold concentration range by
sedimentation velocity analysis, and a 100 fold 
concentration range on gel filtration (the proteins run as 810 kDa, for the
transmembrane domain containing protein, and 480 
kDa for the transmembrane deleted protein).  Equilibrium analysis showed
the presence of a mixture of species for both 
proteins (however, the transmembrane deleted protein clearly showed a
single species by sedimentation velocity?).  We have 
observed aggregation of both proteins over time, which may explain the
equilibrium results. 

What are the potential pitfalls of comparing relative sizes of membrane
proteins in detergent solution by sedimentation velocity 
analysis? 
The data showing the difference in the oligomerization of both proteins are
consistent and very reproducible from different 
patches of proteins. 

We have had trouble finding references for the use of sedimentation
velocity to evaluate s values of membrane proteins in the 
presence of detergents. 

Your help and cooperation is highly appreciated 

Hilal

Hilal Ahmed Al-ashuel
The Scripps Research Institute 
The Skaggs Institute for Chemical Biology 
Mail Drop MB 12, Room MB 28
10550 North Torrey Pines RD
La Jolla ,CA  92037
Tel 619-784-9607
Fax 619-784-9610
Email Hilal@scripps.edu