???? 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, ????, 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
