ºâ½Ü wrote:
> hello all:
> I am a freshman in membrane protein structure study£¬I wonder what
> happen during the process that detergent solubilizes membrane protein
> from bio-membrane ? please guide me to understand this!
> thank you all in advance!
> yours sincerely
> JieHeng from china
>
Hi, ºâ½Ü,
I don't know of any experimental evidence, but what I imagine is this-
As you add more and more detergent, the detergent partitions into the
membrane. The critical micelle concentration is the concentration at
which monomers would be in equilibrium with pure detergent micelles.
At a free detergent concentration slightly lower than that
(because the mole fraction, hence activity, of detergent is lower
in a mixed micelle than in pure detergent micelles), the detergent
partitions massively into the membrane to become the main species
there. Because the size of the head group relative to the tail is
too large to pack nicely in a bilayer, the membrane bulges and breaks
up into round micelles (with greater surface to volume ratio, hence
more room for the headgroups).
These are "mixed micelles", containing everything that was in the
membrane - detergent, lipid, protein. If the amount of detergent
is great enough, membrane components will be diluted out in the
detergent so most micelles will contain zero or one protein
molecule, and these proteins can be purified by conventional protein
purification techniques. The hydrophillic parts of the protein are
sticking out of the micelle. As you purifiy the protein and have
relatively lower detergent concentration in the column buffers,
the micelle shrinks to a narrow belt around the hydrophobic
part of the protein.
If the detergent concentration during solubilization is too
low, many micelles will have two proteins entrapped, and as detergent
is further lost during purification, these become fused together and
difficult to separate- "pseudo-supercomplexes" if the entrapped
proteins are different.
In deciding how much detergent to add, remember it is the *free*
detergent concentration that needs to approach the CMC.
For low CMC deterents and high protein concentrations, the vast
majority of detergent is bound to proteins and partitioned into
membranes, so the total detergent concentration will be many times
the CMC. A good starting point is one gram detergent per gram protein,
plus enough to bring the volume to the CMC.
Ed
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