Saavas and Tommi,
The questions of what is the detergent content of a membrane protein
crystal and how to explicitly determine the amount of detergent in a
crystal are extremely difficult to address. Moreover, is it
worthwhile to even attempt to correct the Matthews coefficient? I
personally don't for a number of reasons. However, one point I would
like to make in this discussion is that ANYTHING concerning micellar
structure or behavior cannot be naively extrapolated to a protein-
detergent complex without firm experimental data. Moreover, when the
protein-detergent complex is in a crystal, it gets even worse. Very
little quantitative work has been done on what is the detergent
structure and behavior in a protein-detergent complex. Peter Timmins
has done the most using neutron diffraction with me and Wolfram Welte
on crystalline systems, as well as in solution (one paper is below).
Pebay-Peyuola, E., Garavito, R.M., Rosenbusch, J.P., Zulauf, M., and
Timmins, P.A. (1995) "Detergent structure in tetragonal crystals of
porin from the outer membrane of E. coli." Structure 3, 1051-1059.
One immediate take home message is that a membrane protein IS NOT in
a micelle, even by definition from surfactant chemistry, nor does a
membrane protein insert into a micelle. In many of the experiments on
detergent binding in surfactant chemistry using styrene beads,
detergent adsorbs onto a hydrophobic surface from single monomer
accretion, and perhaps by micelle fusion. Hence, one should forget
about micelles when talking about a protein-detergent complex. My
rule of thumb from experience is that an "average" membrane protein
of about 50 KD binds about a micelle's worth of detergent, but it
would be a mistake to assume it has all the characteristics of a free
and pure detergent micelle.
Getting back to the amount of detergent in a crystal and the Matthews
coefficient, the detergent layer of protein-detergent complex can
behave like a hard sphere in a crystal or it can fuse with its
neighbors, depending on the detergent used. Changing the detergent
concentration around the crystal, as we do when manipulating a
crystal for many experiments, will change the detergent concentration
in the crystal and can impact the detergent layer of protein-
detergent complex. Thus, efforts to get accurate, detergent-
corrected Matthews coefficients for membrane proteins, may not be
worth worrying about.
Regards,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
****************************************************************
On Sep 24, 2007, at 2:29 AM, Tommi Kajander wrote:
Quoting Edward Berry <[EMAIL PROTECTED]>:
Savvas Savvides wrote:
Indeed, but wouldn't consideration of micelle size affect our
estimation of the number of molecules in the asu, in some cases
significantly?
Good point- I think now that is taken into account by just saying
"membrane proteins tend to have a high solvent content" and taking
that into consideration when you guess the number of molecules.
But it would be nice to account for the detergent explicitly.
Say by analyzing detergent content of the crystals, or in some
ideal cases neutron diffraction with perdeuterated detergent.
with regard to this has anyone actually checked how the micelle
properties
with or without protein "embedded" might differ?? are we assuming
empty
micelle and the protein-added micelle are the same size/Mw? is this
really
so?
--- of course this may further vary depending on the oligomeric
state of the
protein --suppose some neutron scattering studies on model systems
might
give the answer --havent looked. just wondering..
-tommi
--
Tommi Kajander, Ph.D.
Macromolecular X-ray Crystallography
Research Program in Structural Biology and Biophysics
Institute of Biotechnology
PO box 65 (Street address: Viikinkaari 1, 4th floor)
University of Helsinki
FIN-00014 Helsinki, Finland
Tel. +358-9-191 58903
Fax +358-9-191 59940
