Membrane Protein Structure Flashcards
What are the challenges of membrane protein crystallography?
- Flexible and dynamic
- Difficult to recombinantly express
- Hydrophobic surface so aggregate in solution
- Unstable outside of native environment
Why are membrane proteins difficult to recombinantly express?
They require:
- Correct trafficking
- Post-translational modifications
- Membrane insertion
- Correct lipid composition
What are inclusion bodies?
Aggregates of membrane protein in bacteria
If a membrane protein is expressed in E.coli and trafficked incorrectly, what is formed?
Inclusion bodies
How are membrane proteins purified?
- Culture recombinant cells
- Isolate cell membranes through centrifugation
- Extract membrane protein in detergent micelle
- Affinity chromatography
What is critical micelle concentration?
The concentration of detergent above which monomers will spontaneously aggregate into micelles
How are detergents used to purify membrane proteins?
- Detergents shield the hydrophobic surface of the protein
- Detergents form protective micelles around proteins
How are micelle properties tested to check they suit the protein of interest?
Must be determined experimentally
How do you easily screen for suitable detergent conditions?
Make a fusion protein with GFP for sensitive detection
How does Fluorescent size exclusion chromatography for membrane proteins work?
- Make a plasmid of gene with GFP and insert into cells
- Purify membranes and add detergent
- Put extract down membrane column
- GFP allows for production of a unique spectroscopic signal, allowing the behaviour of the protein to be viewed
What is the purpose of fluorescence size exclusion chromatography?
It provides a quick way to screen if your protein is compatible with a detergent
What are the 2 approaches when deciding which proteins to work with?
- Develop understanding and skill so you can get the structure of a specific protein
- Search through lots of proteins non-specifically to find one you can work with
How does a specialist approach to identifying membrane protein structure work?
- Might use recombinant engineering to improve stability
- Undergo alanine-scanning mutagenesis and analysed to see if mutants have greater stability
- Randomly combine stabilizing mutations to get a variant with the highest stability
What is alanine-scanning mutagenesis?
Go through every amino acid and mutate it to alanine
What method is used for finding membrane proteins structures by ‘Brute Force’ ?
High-throughput homologue screening
