Hi,
There are at least 4 methods to try to estimate amount of detergent in a membrane protein crystal which may lead to a more accurate estimation of asu content, etc. At the minimum, this may serve academic curiosity. At the best, if one obtains membrane protein crystals that do not diffract well, any estimates gleaned from this can be used in attempts to improve crystal quality.
These 4 methods are more useful for membrane protein solutions, but if one has sufficiently large crystals, they may be dissolved in buffer and subject to the same experiments.
The techniques are TLC, ANS dye method, DLS (as Savvas mentioned) and FTIR (Fourier Transform Infra Red spec). All of them would require first obtaining a standard curve by first measuring values for buffer solutions with known amounts of detergent, say from 1x to 100x CMC. I have tried all 4 of these methods with several different detergents at different concentration and I think the FTIR comes closest to an accurate measurement. In my experience, DLS readings are difficult when working at or near CMC levels due to aggregation problems.
However, none of the results were totally satisfying, probably due to the complexities of the system (as others have mentioned) which include presence of not only empty micelles along with protein-detergent complex but possibly also lipid-detergent complex and maybe also impurity protein-detergent complexes. Our case was also complicated by the fact that the protein under investigation was a highly elongated molecule and thus DLS estimates of hydrodynamic radius may not have been accurate.
In summary, someone wanting to estimate amount of detergent in their crystals and have sufficiently large and numerous crystals, could try out any of the above methods. The above techniques are quite well documented in literature. The first 3 can be done in-house and so can FTIR. We were lucky enough to have access to an FTIR setup at ALS.
Regards,
Debanu.
--
Debanu Das,
JCSG @ SSRL.
Forwarded message ----------
From: Savvas Savvides <[log in to unmask]>
Date: Sep 24, 2007 3:36 PM
Subject: Re: [ccp4bb] Solvent content of membrane protein crystals
To: [log in to unmask]
I would like to thank Michael Caravito and Gert Van den Berg for
taking the time to share their knowledge and insights on
protein-detergent/micelle complexes.
A series of experiments I carried out using DLS some time ago showed
that the protein-detergent/micelle complex (with LDAO as detergent)
for the membrane protein I was studying had a hydrodynamic radius that
was 20 angs larger than the empty-micelles. The experiments were done
in the protein storage buffer: 20 mM Tris 8.0, 250 mM NaCl, 0.15 %
LDAO, which is far from a crystallization condition!
As Michael and Gert pointed out, it is a risky business to
draw quantitative conclusions from such measurements due to the
effects of so many factors. Nonetheless, it was quite
informative to me at the time to see a marked difference between the
protein-detergent complex and the empty-micelles. Aside form bringing
home the concept that the interaction of the protein with
detergent/micelle had a true physical meaning that could be translated
to particle augmentation, it also helped me benchmark my
gel-filtration runs.
Best regards
Savvas
----
Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [log in to unmask]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html
Quoting "R.M. Garavito" <[log in to unmask]>:
> 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: [log in to unmask]
> ****************************************************************
>
>
> On Sep 24, 2007, at 2:29 AM, Tommi Kajander wrote:
>
>> Quoting Edward Berry <[log in to unmask]>:
>>
>>> 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
>>
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