[RASMB] questions from a membrane newbie

[Tom Laue]

Hi Chad-
  Optical reasons underly the caution/prohibition against using IF 
optics with detergent-containing solutions. The IF optics are sensitive, 
to the 6th decimal place, to the difference in refractive index between 
sample and solution. Since every component in the solution contributes 
to the refractive index (not entirely equally, but significantly), very 
small mismatches in concentrations will result in an inaccurate 
assumption that the observed fringe displacement is entirely due to the 
protein. While it is also essential to dialyze samples for thermodynamic 
reasons, the error incurred in small unless one is trying to extract 
nonideality information (when it can confound interpretation).
  In your case, you may have dialyzed your sample (C8E5 is a dialyzable 
detergent), in which case IF will be fine. Also, you carefully matched 
the buoyancy of the detergent with the solvent. So long as the vbar of 
the detergent is insensitive to detergent concentration (which amounts 
to assuming the detergent monomer and detergent micelle have the same 
vbar), there will be little or no contribution to the IF signal since 
there is no detergent gradient. If you did not dialyze the sample, then 
you must have had a very well matched buoyancy and got lucky.
Best wishes,
Tom

Chad Brautigam wrote:
> 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
>
>
>
>
>
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb
>

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Rudman Hall 379
46 College Road
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
www.camis.unh.edu
www.bitc.unh.edu