Nxsib:community/ill membrane bag

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ILL Membrane Bag

This page serves is set up as a community site for the ILL 'Membranes' Block Allocation Group (BAG) on the D22 Small Angle Neutron Scattering instrument. Information here refers to the sample production and experimental set up for experiments that are part of this BAG. Feel free to add information about your samples and experiments as well as any useful tips or advice. It may be possible to use a calendar to aid in scheduling for the group.

Logistics

A discussion with the chair of the ISIS Large Scale Structures assessment panel has suggested that a proposal from the BAG group (or a part of it) to do preliminary experiments on LOQ would be considered favourably. This may well be worth considering for the next ISIS round.


BAG group experiments

The Structure of Membrane Proteins from Small Angle Neutron Scattering: The Purple Bacterial Reaction Centre

Neylon, Hughes, Heenan, Jones, Malfois - The BAG proposal can be viewed here

Our aim is to develop the techniques for sample production and background subtraction to improve the structural data that can be obtained on membrane proteins. Our first target is developing optimal mixtures of part and fully deuterated lipids that provide lipid vesicles where both tails and headgroups have the same SLD making it easier to match them out. We are using the 'Rhodobacter sphaeroides' Reaction Centre as a model system as it readily produced, a crystal structure is available, and it has a distinct absorbance spectrum that is sensitive to degradation making it possible both to ensure that the protein is intact after reconstitution into lipid membranes and to accurately determine the concentration.

Sample preparation

The protein is exchanged from LDAO detergent to beta octyl glucoside on a DEAE column. The protein in bOG micelles is then dialysed against buffer with DMPC vesicles (1 mg/mL) overnight. The reconstituted protelipid vesicles were then dialysed to the appropriate D2O/H2O ration and concentrated to ~10 mg/ml nominal lipid concentration using spin filters and re-extruded. There was no significant loss of protein absorbance through this process.

For the most recent experiment only D54 (tail deuterated) and D13 (head deuterated) DMPC were available. This limited the D2O content that could be used for matching to around 65%.

Current results

The contrast matching seemed to work well with the matchpoint being very close to that predicted. Depending on the background subtraction applied there was either no scattering above a buffer base line or a small amount at low Q for ~10mg/mL 200 nm vesicles. In the protein-lipid samples scattering above the background was observed that was dependent on the concentration of protein. However the scattering is not consistent with the size or scattering profile predicted from the crystal structure and suggests aggregates at least 2-4 times the size of the monomeric protein. This is similar to what Stephan sees for MscL in his experiments. In addition signal strength was weak due to the lack of contrast between the protein and the solvent.

Future experiments

In the future this will be improved by using lipid mixtures that can be matched at higher D2O concentration and ultimately by deuterating the protein. We will also look at scattering from the protein in detergent micelles. We also need to comprehensively look at the different mixed lipid compositions to identify the quality of the contrast matching in practise. This could probably be better done on LOQ at ISIS rather than using up the D22 time.