[RASMB] questions from a membrane newbie

[Chad Brautigam]

Hi, All,

So, I just did my first SE experiment of a membrane protein in the  
presence of detergent.  The object:  determine the oligomeric state of  
the protein in question.

I used C8E5 and matched the density of the buffer to the density of  
the detergent empirically, ala Karen Fleming (Burgess et al., Meth.  
Cell Biol., vol. 84, 181).  I did five different concentrations of the  
protein, which was solubilized in a buffer containing 2X the CMC of  
the detergent, and collected 250 and 280 nm data.  I used three  
different rotor speeds, the last featuring significant meniscus  
depletion.  I used long columns (this is a very well-behaved protein).

The global fits from SEDPHAT look pretty great, and I am pleased with  
the mass result, i.e. it is near to nx, where n is the stoichiometry  
and x is the mass of the monomer.  So far, so good.

But, upon perusing the literature, I came across a very interesting  
and categorical statement in le Maire et al. (2008), Nat. Prot., vol.  
3, pp. 1782-1795:  "...interference optics may not be used" when  
trying to "evaluate molecular masses and the composition of the  
detergent-solubilized complex."  Those quotes are a little rearranged,  
but I think I've not altered their meaning.

Why, you ask, is that an interesting statement?  Because, dear  
hydrodynamicist, in my naivete, I collected the IF data as well.  Now,  
I was sort of careful about it, meaning I mechanically "aged" the  
cells by spinning them at 50,000 rpm, torquing, spinning, torquing,  
etc., until no movement of the torque wrench was observed upon  
torquing.  This protocol is suggested in the protocol at www.analyticalultracentrifugation.com/SEprotocols.htm 
.  I also urged my collaborator to match the buffers in the reference  
and sample cells well. We matched the menisci as well as we could.   
But, because we were a bit short on time, I didn't measure the water  
blanks, instead relying on the program (SEDPHAT) to calculate RI and  
TI noise elements as in Vistica et al. (2004) Anal. Biochem. pp 234-256.

I mention all of this because I was able to get a *beautiful* fit from  
the IF data.  The mass was almost exactly that measured in the ABS  
experiments.  Indeed, I can include the IF & ABS data in a global  
analysis and it works very nicely, i.e. I get virtually the same mass  
in the result.

So, finally, to my question.  Why did this work, given the admonition  
of le Maire et al.?  Am I misreading that (excellent) paper?  Have I  
executed a flagrant violation of the laws of physics?  If so, I stand  
ready to accept my punishment.

An adjunct question:
The signal increment in IF that I refine is much higher than I would  
expect for this protein.  Does that mean that I am measuring the  
signal increment of protein + bound detergent?

Thanks in advance for any help,

Chad

======================================
Chad A. Brautigam, Ph.D.
Assistant Professor
Department of Biochemistry
The University of Texas
Southwestern Medical Center at Dallas
Department of Biochemistry
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
Office:  214-645-6384
Fax:  214-645-6353
Email:  chad.brautigam at utsouthwestern.edu