Lecture 2: Detergents
What makes detergents distinct from membrane lipids?
1) They are usually synthetic molecules, not seen often in nature. 2) Usually have a single hydrophobic tail as opposed to two. 3) They form micelles in solution not bilayers.
What is a detergent?
A compound that dissolves hydrophobic molecules into aqueous solution. They are amphiphilic because they have a hydrophobic tail and a hydrophilic head group.
What are Multilamellar vesicles (MLV)? How big are they? What are they good for?
A suspension formed by many membrane lipids when they are simply dispersed in water after being totally dry. They are basically a Russian nesting doll of lipid bilayers. (That's twice in 12 hours I've used the term Russian nesting doll and I'm not sure how I feel about that.) They are large enough to scatter light and make the solution look milky a lot of the time. They are pretty big, ~1-10 uM (10uM is the limit of visibility). They are good for measuring the physical properties of membranes.
What is the average molecular weight for a micelle?
Between 15-100 Kda so about the size of your average protein in terms of mass. (Except for bile salts which are small: ~ 1-4 Kda)
Do membrane lipids have a CMC?
In theory yes they do, but in practice they don't because it is so small we aren't even able to measure it. So not really even worth thinking about it.
Which are more denaturing: Ionic or nonionic detergents? Give an example or two of each.
Ionic are more denaturing, an example would be SDS. An example of nonionic would be Triton X-100 or Tween-20.
What is a Triton Phase partition assay?
It is when you take a solution with lipid membranes containing membrane proteins you are interested in and then dissolve the membrane with triton. After dissolving with triton the hydrophobic membrane proteins with hang out in the micelles that have formed. Then you send the temperature of your solution up to the cloud point of triton and get it high enough to cause phase separation. Your membrane protein should now be in your detergent phase and can be separated from the stuff in the boring old liquid stage. This is used as a purification technique, but it can also be used to identify whether or not your protein of interest is in the membrane or not.
What is the difference between lipoproteins and lipid droplets?
Not all that much. Lipid droplets store cholesterol and triglycerides inside the cytoplasm of a cell using a phospholipid monolayer. Lipoproteins also balls of phospholipids with dissolved centers of cholesterol, cholesterol esters and triglycerides BUT these travel through the blood stream. Both have proteins in/on them.
What the hell are lysolipids?
Phospholipids that are missing one acyl chain. Which is really similar to a detergent but whatever I'm not the expert here I'm just writing what he tells me.
What are the two classes of nonionic detergents?
Polyoxyethylenes and Alkyl Glycosides
What is the cloud point of a detergent?
The temperature above which some non-ionic detergents become "cloudy" due to micelle aggregation. The cloudy appearance is due to the micelles getting so large that they are scattering light now. At high enough temps the aggregates form a detergent rich phase at the bottom.
What are Bicelles?
These are formed when you have really short acyl chain lipids and normal length lipids, basically you form a membrane with the long ones and then the little ones act to seal up the sides and stuff. Apparently they're useful in NMR.
What are the biological detergents and what are they used for?
These are the bile salts and they too are used to dissolve hydrophobic things in the body like fats you eat. They are derived from cholesterol.
What are low CMC detergents good for? What are they bad for?
They are good for dissolving membranes, but really hard to remove so they're bad for reconstitution processes.
What are Small Unilamellar vesicles (SUV)? How big are they? What are they good for?
They are just really little spheres of membrane lipids that trap a teenie-weenie bit of water. They are so GD curved that 2/3 of their lipids are on the outer leaflet and 1/3 are on the inner. They are ~250A in diameter which is the smallest these can possibly get and still be closed structures. There are usually ~5000 lipids per vesicle. They are good for uhm idk he didn't really say they're good for anything. You can form them through sonication of larger vesicles though! They're really hard to pellet out via centrifugation and can sometimes be formed through dialysis of mixtures with lipids and the detergent cholate.
What is the Critical Micelle Temperature (CMT)?
This CMT is the temperature bellow which a detergent forms an insoluble solid rather than a micelle. It basically freezes and precipitates out of solution.
What is an emulsion? Name two types we talked about.
This is a particle with a dissolved hydrophobic core and a hydrophilic outer shell. We talked about Lipoproteins both high and low density and we talked about lipid droplets!
What is the way we describe Micelle size?
This is given by the aggregation number. The aggregation number is defined as the number of detergent molecules per micelle. Alternatively, if you want to be unnecessarily complicated it can be defined as: (MW of micelle) / (MW detergent monomer)
Describe the critical micelle concentration (CMC).
This is the concentration at which micelles first appear. Think of it as the solubility of the detergent molecule! Once you get to a high enough concentration of detergent monomers in solution you effectively saturate it with monomers. Then when you start adding even more than this they start to "precipitate out" in the form of micelles. The concentration at which you get this "precipitation" of detergent monomers into micelles is the critical micelle concentration.
What is membrane reconstitution?
This is when you reassemble a membrane from lipids and membrane proteins by mixing the original membrane with detergent to break it down into small pieces (mixed micelles). Then you remove said detergent via dialysis or some other method and allow the original to reform.
What is the reason we use detergents in gel electrophoresis? What is important to note about hydrophobic proteins in this process?
We use them as a way to denature proteins to then run in a gel to determine the weight of the protein. Important to note that membrane proteins are so hydrophobic on their surfaces that they will not open up as much as other proteins upon denaturation and this can cause them to run fast on the gel.
How do you determine the properties of a mixed micelle?
You basically don't. A ton goes into it including composition, percentage of each component, temperature, proteins within the membrane and shit like that.