Jacob,
I doubt that the spherulites formed in the presence of detergents are
"primary" detergent phenomenon. However, detergent micelles and the
protein-detergent aggregates (please don't say protein-detergent
micelles) will condense (phase) out in the presence of high salt or
polymers to form droplets. What you have to remember about these
droplets is (1) the membrane protein concentration is markedly
increased (up to 100-200 mg/mL or into the mM range) and (2) the
detergent concentration also increases (10-100x).
The first situation with a membrane protein can lead to spontaneous,
but uncontrolled nucleation that can lead to microcrystal or
paracrystal formation of the protein. Hence, spherulites form from
the droplets.
In the second situation, the detergent concentration can also cause
the system to reach one of the more ordered mesophases. While for
most nonionic and zwitterionic detergents, the concentrations we use
are well below these mesophases in all cases. However, we mix these
detergents with all sorts of other components (salts, PEGs, MPD,
etc.) which can markedly alter the phase behavior of the detergent.
Thus, droplets of highly concentrated detergent in the presence of
crystallization reagents could be induced to form liquid crystals,
which leads to spherulite formation.
As virtually all of the published phase diagrams for our detergents
of interest are of binary (detergent and water) and ternary
(detergent, low salt, and water) systems, they are useless for
predicting what is happening. Moreover, there is the dreaded specter
of contamination of your detergent. For example, alpha-octyl
glucoside crystallizes quite easily, while the beta anomer does not
under almost all conditions. Although most commercial sources of
beta-octyl glucoside are pretty pure (~98-99% pure), the detergent-
rich droplets could be enriched in the unwanted alpha contaminant.
Similar paracrystalline structures can be seen with contaminants of
other nonionic and zwitterionic detergents.
My advice to you is to see if the same phenomenon occurs in protein-
free controls drops. Remember that your membrane protein is a
"perturbant" of detergent phase behavior, and the protein-detergent
aggregate will also have its own phase behavior. If spherulite
formation ONLY occurs in the presence of your membrane protein, then
you could argue that the spherulites are made up of poorly
crystallizing membrane protein. Not completely a positive result,
but it is something.
Sadly, there are no papers about this. However, the Crystals page of
the Kay Diederichs' CCP4wiki (http://strucbio.biologie.uni-
konstanz.de/ccp4wiki/index.php/Crystals) now has a section for hints,
tips, & observations about crystals ("I have crystals, but are they
salt?"). I would encourage people to post their insights there.
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]
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On May 22, 2008, at 8:58 PM, Jacob Keller wrote:
Sorry for this non-CCP4 question, but I know no better venue to ask
this:
Do people see detergent spherulites or other artifacts in crystal
screens in the presence of dodecyl-maltoside or other detergents?
Are there any papers about this? I have seen some papers talking
about the relationships between salt, temp, cmc, and cloud points,
but nothing on precisely this topic (detergent-related
crystallization artifacts).
Best Regards,
Jacob Keller
ps. in passing, it seems like it would be a great idea to get
together an excel database of all false-positive results (e.g.
phosphate salt crystals) commonly found in the usual crystal
screens. One could then search it to see whether one's current
crystallization conditions have be villified in the past.
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: [EMAIL PROTECTED]
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