Lecture 7: Difficulties of Obtaining Membrane Protein Structures Flashcards
Why is it difficult to obtain 3D crystals of membrane proteins? Give 5 reasons
1) needs lots of pure protein - can recombinantly express proteins to get them in high amounts
2) proteins need to be removed from the membrane through dissolving in detergent
3) hydrophobic stretches associate in a non-specific way and precipitate
4) need regular contacts between proteins in the crystal
5) over-expressing membrane proteins will often kill the cells you are expressing them in.
Why is the choice of host important for expression?
Need to express genes in like cells ie, human gene is unlikely to work in bacteria
How are membrane proteins usually extracted from the membrane?
By detergent, purified to homogeneity
What happens at a certain concentration of detergent?
You get mixed micelles forming
What form of purification is usually used for membrane proteins?
Affinity purification
Describe how the structure of LacY was obtained and the problems faced
Ron Kaback used X-ray crystallography. The structure was a mutated inactive form that is inward facing. Had to reduce the flexibility to get crystals. Problem as many membrane proteins need flexibility to function.
Describe the problems faced after GLUT1 structure was solved in 2-14
Structure was solved for a locked conformation. Doesn’t give full mechanistic detail as doesn’t show the other conformation. Could crystallise in the other conformation but need to get mutants in this conformation.
What is a Fab fragment? What happens when they form a crystal?
Antibody with the long part of the heavy chains removed. All of the Fab fragments aggregate in the crystal.
What are the three tricks for crystallisation?
Use a thermostabilised protein eg fusion with T4 lysosyme makes proteins less flexible.
Use an antibody or Fab binding
Use of detergent: short chain detergents form micelles where the hydrophilic regions can form crystal contacts. Long chain detergents form large micelles with the proteins inside so hydrophilic regions won’t be able to reach out so makes good crystals.
What are the majority of structures solved with? What is the problem?
X ray crystallography. Difficult for membrane proteins
What is needed for electron crystallography and what was solved by this?
2D crystals (a sheet of membrane). Bacteriohodopsin.
How is a 3D map built from 2D crystals?
Get diffraction pattern as electron beam passes through then tilt the sheet to get a tilt series and build 3D map.
What is atomic force microscopy?
Low resolution structures obtained. get very good data with 2D crystals when sheet is perpendicular.
How is cryo-EM carried out?
Sample is purified in detergent, put into a grid and frozen. View on electron microscope. Pick particles of same orientation. Combining the images builds up a 3D map and model. There is class averaging and each class average represents a different view of the particle.
What are the advantages of cryo-EM over crystallography?
No crystals needed
Reduced pure protein requirements
Multiple structural states may be present in the same sample and may be separated computationally
Proteins can be in detergent solution
By looking at different structural states, may be able to deduce something about the mechanism of action
