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| Structure of a micelle. Taken from http://en.wikipedia.org/wiki/File:Micelle.png |
This great paper is on the Chemistry-Biology interface. The
group has developed a novel fluorinated (rightfully a ‘fluorous’) detergent
that can find potential applications in membrane biology. Membrane proteins are
biologically important, they serve as many drug targets, and infamously
difficult to deal with. Detergents are often used to solubilize the membrane
proteins so that they could be manipulated for further studies. Detergents are amphiphiles, which means they have both a
polar and a non-polar part, so that they can interact with the phospholipid and
thereby disrupt the membrane bilayer structure.
As I have said, a fluorous compound does not necessarily
like the 'fatty' organic layer, so a 2-phase system can be generated
potentially. However, traditional wisdom suggests that although fluorinated
compounds don't like aqueous phase too, that does not make them good
detergents. The reason is two fold - first, the fact that fluorous and organic
are not miscible means that it can be hard for fluorous compounds to interact
with the organic membranes. And fluorine atoms are large compared to hydrogen
atoms, so sterically it can be tough for them to mingle with the membranes. So,
fluorous compounds are considered detergent-resistant, and they are less likely
as candidates of great detergents. That is not the end of the world - because if
there exists a ‘fluorinated detergent’, then its fluorinated tail will be
unlikely to interact with the hydrocarbon part of the membrane, and then the
protein-membrane properties will not be affected and the integrity of the
membrane protein will be restored. This paper just shows one of these cases.
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| The structure of F6OM, the detergent the researchers developed, and F6OPC, another detergent to compare with. Taken from [1]. |
The researchers have synthesized a novel fluorinated
detergent known as F6OM, which consists of a carbohydrate end and a fluorous
end (C6F13). They have compared F6OM with another fluorinated
detergent known as F6OPC, to show how different their properties can be. While
F6OPC also consists of a C6F13 terminal, it also consists
of a polar, and indeed zwitterionic, end - a cationic ammonium and an anionic
phosphate here. The respective self-assemblies of the 2 contenders are very
different - F6OM appears as long rods but F6OPC looks like small spheres.
By using light scattering experiment, the group discovers
that micellar F6OM can solubilize with a
derivative of phosphocholine (POPC), and this solubilization can be enhanced by
increasing the F6OM to a certain concentration, or increasing the temperature. This
is not the case for the counterpart
F6OPC. The observations agree to conventional understanding – a higher
temperature should encourage membrane destabilization and a faster detergent
translocation. The researchers carry out further isothermal titration
calorimetry (ITC) to develop a quantitative understanding of the phenomenon. Furthermore,
they can establish a phase equilibrium of F6OM, as present in a bilayer or a
micelle. At a medium concentration, both types are seen to co-exist. One thing
I would like to point you to is the use of
31P and 19F NMR in this work. The dynamic upfield shift of CF3
signal in 19F NMR signifies solubilization.
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| The SDS-Page analysis for refolding experiment. When the concentration of F6OM reaches a certain level, folded protein can be observed. This is not so in the case for F6OPC, even when its concentration is raised to the same level as F6OM. Taken from [1]. |
An interesting aspect of membrane biology is the study of
unfolding / refolding of proteins and membranes, in a sense you are building
(or reconstituting) the membrane architecture from scratch. Our F6OM turns out
to be a great candidate as a chaperone for this. By adding a CH2
spacer between the fluorous group and the carbohydrate section, the analogue shows
promise. First, this analogue has a self-assembly to make it appear like a
membrane bilayer, and then a phospholipase (OmpLA) can be refolded, and the
whole protein-membrane complex becomes a functional proteolipase. The SDS-Page
analysis shows that when the concentration of
F6OM reaches a certain level, the phospholipase can be refolded into an
active state, and this seems to out-rival the other contender, F6OPC.
I think this is a great paper that illustrates the use of a
chemical compound for biological applications. There are a lot of nice
techniques inside – electron microscopy, light scattering, ITC, Fluorescence Spectroscopy,
NMR and various assays. I am aware of some of them but I do not have practical
experiences on these for my work, so I have learnt a lot from this paper. I
encourage you to read more about those techniques, especially if you are
working in biochemistry / chemical biology!
by Ed Law
27/03/2015
Reference:
1. A Fluorinated Detergent for Membrane-Protein Applications E. Frotscher, B. Danielczak, C. Vargas, A. Meister, G.
Durand, S. Keller.
DOI: 10.1002/anie.201412359