Membrane Proteins 3

Question 1

What is the overall procedure of structure determination of MPs by crystallography?

Answer 1

Recombinant expression followed by solubilisation (membrane extraction), purification and crystallisation.

Question 2

What is the clinical relevance of structure determination of MPs?

Answer 2

85% of drugs act via membrane proteins. MPs important in many disease states.

Question 3

Why are bioreactor vessels to express recombinant membrane proteins?

Answer 3

To produce large scale cultures under controlled conditions. This is important as it is often difficult to express enough of a membrane protein (may be toxic to the cell).

Question 4

What systems are best for expressing human MPs?

Answer 4

Mammalian/insect/baculovirus systems.

Question 5

What are some of the problems associated with expression?

Answer 5

May get non-specific degradation (host cell proteases), removal of his tag, formation of inclusion bodies (may be able to refold) and expression of a non-functional protein.

Question 6

What proteins has refolding of inclusion bodies been successful for?

Answer 6

beta barrel proteins

Question 7

How does expression of a non-functional protein arise?

Answer 7

May be missing a lipid/PTM or may be incorrectly localised within the cell.

Question 8

Why can detergents be used for membrane extraction?

Answer 8

Have the same dual hydrophobicity as the membrane.

Question 9

What happens to detergents at their critical micelle concentration (High conc.)?

Answer 9

Spontaneously associate to form micelles- head groups exposed, tails shielded in micelle core.

Question 10

How are protein-detergent complexes formed and why are they useful for structural determination?

Answer 10

In the micelle state, the detergent can interrupt hydrophobic interactions between MPs and the phospholipid bilayer. Detergent displaces membrane to form protein-detergent complexes- useful as are soluble in solution.

Question 11

What are the problems associated with solubilisation?

Answer 11

May require many trial and error experiments. Recovery low- 30-40% is considered good.

Question 12

How are membrane proteins typically purified?

Answer 12

His tag affinity chromatography followed by size exclusion chromatography. MPs must be kept in detergent throughout.

Question 13

Why can’t more than 2 purification methods be performed?

Answer 13

MPs form stronger non-specific interactions with the media which means protein is lost at each step. Lipids are also lost as they form strong interactions with the media- destabilisation of micelle.

Question 14

What is the method of solubilisation?

Answer 14

1. Lysis and centrifugation to pellet membranes.
2. Add detergent, incubate at 4 degrees for 30 mins- 1hr
3. High speed centrifugation to remove insoluble proteins and lipids.
4. Harvest soluble material.

Question 15

What occurs after purification?

Answer 15

The pure protein is concentrated using a molecular weight cut off filter and exchanged into a low salt buffer. Purity of sample assessed by SDS-PAGE. Protein identity confirmed by either mass spec or N term seq.

Question 16

Why can the band for MPs be seen at an incorrect Mw on SDS-PAGE gel?

Answer 16

SDS doesn’t completely disrupt interactions within the membrane protein due to high hydrophobicity- run quicker than expected. Otherwise there may have been degradation or an incorrect protein may have been purified.

Question 17

How can most membrane proteins be crystallised?

Answer 17

Via vapour diffusion

Question 18

Why is the formation of a crystal lattice less likely for MPs?

Answer 18

Belt of detergent means they pack less closely. Therefore form weaker crystal contacts and lattice formation less likely. Can only form crystal contacts using hydrophilic regions (normally small). Detergent shields the hydrophobic regions.

Question 19

How does poor crystal formation affect crystallisation data for MPs?

Answer 19

Lattice is likely to contain mistakes and be small- will diffract less X-Rays than a crystal produced from a soluble protein. This results in low data quality.

Question 20

How can crystallisation of membrane proteins be improved?

Answer 20

Decreasing size of micelle- larger region available for crystal contacts. Increasing size of protein- more regions available for crystal contacts. Engineer- to be more stable in solution, then allows micelle size decrease.

Question 21

Name some commonly used detergents.

Answer 21

OG (C8) and DDM (C12)

Question 22

What is the advantage of using OG?

Answer 22

Forms small micelles - single head group, 8 carbon tail.

Question 23

What is a disadvantage of using OG?

Answer 23

MPs have more exposed regions- more likely to form non-specific interactions, crystal more likely to degrade.

Question 24

Describe DDM.

Answer 24

Disaccharide head, C12 tail. Forms large micelles. Maintains most membrane proteins- suitable for solubilisation, purification and crystallisation.

Question 25

How is detergent size decreased?

Answer 25

Exchange from a larger detergent to a smaller detergent. Can generate mixed micelles, add DDM and a small amount of OG- slightly shrinks micelle. Can also be decreased via the addition of small amphiphiles.

Question 26

What is the advantages of stabilising detergents?

Answer 26

More effective at maintaining stable MPs in solution. Target protein less likely to denature and more likely to retain function.

Question 27

Describe MNG-3.

Answer 27

Stabilising detergent. Double disaccharide head, double C12 tail connected by central C- very rigid, forms stable micelle.

Question 28

What are the main difficulties when working with membrane proteins?

Answer 28

Naturally low expression. Naturally located within the lipid bilayer. Difficult to keep stable enough to work with.

Question 29

When is lipid cubic phase crystallography used?

Answer 29

As an alternative to vapour diffusion- useful for large MP-detergent micelles. Particularly successful for GPCRs.

Question 30

Describe lipid cubic phase crystallography.

Answer 30

Protein sample mixed with lipid phase and the protein is exchanged out of the detergent micelle into the lipid phase. Protein molecules interact with each other and form crystals within the lipid matrix. Takes a while for crystals to grow- must use MNG-3 to keep protein stable.

Question 31

Why may it be useful to add an antibody fragment to the target protein?

Answer 31

May increase the size of the hydrophilic region available and mediate crystal contacts.

Question 32

Describe types of protein engineering that may increase the likelihood of crystal formation.

Answer 32

Removal of flexible regions- more ordered crystals. Addition of soluble domains (T4L) - more hydrophilic surface available. Mutation of hydrophobic surface residues- more hydrophilic region available. Alanine scanning- find most stable combination of mutations.

Question 33

Why is the synchrotron beneficial to crystallography of MPs?

Answer 33

Can focus X-Ray onto small MP crystals. Allows targeting of small regions on small crystals- to get regions of good diffraction data. Can be combined to give a fully dataset.

Question 34

What can be done if it was not possible to obtain large enough crystals?

Answer 34

Datasets from multiple smaller crystals can be combined and built up to resolve the structure for the overall molecule.