Membrane Protein Structure

Question 1

What are the challenges of membrane protein crystallography?

Answer 1

• Flexible and dynamic
• Difficult to recombinantly express
• Hydrophobic surface so aggregate in solution
• Unstable outside of native environment

Question 2

Why are membrane proteins difficult to recombinantly express?

Answer 2

They require:

• Correct trafficking
• Post-translational modifications
• Membrane insertion
• Correct lipid composition

Question 3

What are inclusion bodies?

Answer 3

Aggregates of membrane protein in bacteria

Question 4

If a membrane protein is expressed in E.coli and trafficked incorrectly, what is formed?

Answer 4

Inclusion bodies

Question 5

How are membrane proteins purified?

Answer 5

• Culture recombinant cells
• Isolate cell membranes through centrifugation
• Extract membrane protein in detergent micelle
• Affinity chromatography

Question 6

What is critical micelle concentration?

Answer 6

The concentration of detergent above which monomers will spontaneously aggregate into micelles

Question 7

How are detergents used to purify membrane proteins?

Answer 7

• Detergents shield the hydrophobic surface of the protein
• Detergents form protective micelles around proteins

Question 8

How are micelle properties tested to check they suit the protein of interest?

Answer 8

Must be determined experimentally

Question 9

How do you easily screen for suitable detergent conditions?

Answer 9

Make a fusion protein with GFP for sensitive detection

Question 10

How does Fluorescent size exclusion chromatography for membrane proteins work?

Answer 10

• 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

Question 11

What is the purpose of fluorescence size exclusion chromatography?

Answer 11

It provides a quick way to screen if your protein is compatible with a detergent

Question 12

What are the 2 approaches when deciding which proteins to work with?

Answer 12

• 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

Question 13

How does a specialist approach to identifying membrane protein structure work?

Answer 13

• 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

Question 14

What is alanine-scanning mutagenesis?

Answer 14

Go through every amino acid and mutate it to alanine

Question 15

What method is used for finding membrane proteins structures by ‘Brute Force’ ?

Answer 15

High-throughput homologue screening

Question 16

How does high-throughput homologue screening work?

Answer 16

• Try and express proteins using careful design
• Purify successfully expressed proteins
• Crystallise purified proteins looking at monodispensity and thermal stability
• Optimise crystallisation
• Collect scatter patterns and determine structure

Question 17

What is monodispensity?

Answer 17

Getting a single nice peak on a size exclusion chromatogram

Question 18

What can help optimise crystallisation for membrane proteins?

Answer 18

• Detergent type (which may be different to the one used for purification)
• Detergent concentration
• Antibodies