Techniques in membrane biochemistry Flashcards
Why do we care about membranes?
- membranes are barriers
- control movement of substances in and out
- control the flow of information
- capture and release of energy
What is a liposome?
a spherical sac of phospholipid molecules enclosing a water droplet,
Name 4 forms of liposomes. (and which 2 are the most wanted)
MLV, MVV, LUV & SUV (latter 2 the most wanted)
How do you control liposome formation?
- start off with a white powder
- mix - solubilise in chloroform to be able to mix all elements
- Dry in a vacuum
- Re-suspend in aqueous buffer
- left with MVV’s and MLV’s - Extrude if you want LUV/SUV
- Sonication occurs if you want SUV’s
- rise temp above the phase transition
- allows for definition
How does differential centrifugation work?
- Resuspend sucrose in 0.25m sucrose buffer in homogeniser
- Isotonic
- Stops things from mixing
- Push sample around the sides
- Sheers cells open gently (no bubbles) - Different organelles at each centrifugation
- Based on density not mass
- Isopycnic separation
- After each separation, there is an increase in the speed
How does density gradient centrifugation work?
- method of separation based on the density of the species
- isopycnic separation
1) Prep test tube with different density layers
2) Organelle fraction at the top
3) Centrifuge overnight at a high speed
4) Organelles will separate & show different layers - Protein markers will allow you to define/ identify components
How does solvent extraction work?
- mix well
- filter to get rid of anything extra
- add salt solute
- allow to separate
- extract the chloroform layer
What is a mild extraction of proteins method?
- changes in ionic strength
- changes in pH
- addition of EDTA (metal chelators)
What is a harsh extraction of proteins method?
- chaotropic agents (agents that disrupt H-bonding) such as Urea
(not too much as protein will unfold)
Why can you not use solvents to extract proteins off a membrane?
Solvents will kill the protein
How do you chose which detergent to use for a protein?
Have to chose the detergent concentration above the CMC conc to solubilise integral membrane proteins.
How do you use a detergent to solubilise an integral membrane protein?
Mix detergent with the membrane protein
- Tag
- once solubilised, identify the lipid mixed micelle and remove
What are amphipols?
Short amphipathic polymers that can substitute for detergents to keep integral membrane proteins water soluble
How do you use a amphipols to solubilise an integral membrane protein?
Mix amphipols with the membrane protein
- Tag
- once solubilised, identify the disc created
What causes separation in TLC?
Attraction between compound and solvent and compound and stationary phase.
What is the stationary phase in TLC?
The silica gel
How does Gas liquid chromatography work?
- Inject gas into columns
- The column’s are silica coated non volatile
- Detector at the end will detect when different fatty acids will come off the column, recording retention times.
- the longer the fatty acyl chains or degree of un-saturation, the longer the retention times.
How does Gas chromatography Mass Spectrometry work?
The same as GLC but with mass spec after the detector
How does 2D TLC work?
- Disrupts the outer membrane
- Really good for lipids
- No info on chain length but info on head group
How does fluorescence work?
- electrons are excited
Jumps from a low energy level to a high energy level - Some energy is lost through vibrations
- Through internal vibrations, it will jump from one of the vibration states onto another
- Can jump back through internal conversion such as heat or via fluorescence
○ Wavelength used to get back is longer as some of it has been lost
○ Makes it very sensitive as no contaminating light
○ Occurs in molecules with double bonds and found in aromatic molecules
How does a basic fluorescence spectrometer work?
the source and detector are at right angles of sample, so all exciting light goes straight through
How does polarity change emission of fluorescence?
- in more polar environment, the lower the emission but the higher the quantum yield
How can you test the phase transition temperature of a membrane?
- below phase transition in the gel phase, NPN (a fluorescent lipid probe) cannot enter into where needed due to the alignment
- as temp increases and phase transition changes, NPN can get in
How is the fluorophore re-orientated?
- At Zero Kelvin, the fluorophore will be excited with polarised light. Same energy released as put in, maximum emission
- At Room Temp, the fluorophore excited with polarised light will move before the photon is emitted
- tumbling of molecule
- some of the polarisation is lost and therefore the signal is weak
How can you use the orientation of the fluorophore as a measure of when the Tm is?
- the fluorophore can orientate itself within the membrane
- when in the gel phase it orientates to the alignment of the bilayer
- as it goes ABOVE the phase transition temp, orientation is lost and tumbling can occur, leading to a weaker signal
- monitoring the depolarisation of the emitted light as a function of time reveals the average angular displacement of the fluorophore between the time of excitation to the time of emission
What is intrinsic fluorescence?
- using whats in the protein that causes fluorescence such as Tyr and Trp
- allows you to study the protein without affecting the protein
How are solvent effects used for studying membrane protein function?
- measure stability of proteins when unfolded
- fluorescent emissions shifts when unfolded
if polar environment: - fluorescence moves to RED end
- becomes LESS intense
if non-polar environment: - fluorescence moves to BLUE end
- becomes MORE intense
How does FRET work?
- the emission of one fluorophore excites the fluorophore of the one next to it
- energy from first fluorophore is transferred non-radiatively
- wavelength of the fluorophore 1 is longer
- don’t see emission of 1 but see emission 2 so you know FRET has occured
What distance do the two fluorophores need to be to achieve 50% transfer efficiency?
3nm - 7nm
What can FRET be used for?
- identifying the things in the local environment
- protein lipid selectively
What can be observed in nuclear magnetic resonance?
- only isotopes with an odd number of protons and/or neutrons can be observed
- e.g. H1, C13, N15
How does nuclear magnetic resonance (NMR) work?
- atom will align with magnetic field or don’t align
- change in energy is related to the frequency of the electromagnetic radiation that is pulsed at the sample
Why should the proton nuclei in different compounds behave differently in the NMR experiment?
- Not all the H give the same signal
- this is due to where the nucleus is, the electron shields the nucleus and affects how it behaves in the magnet
- can separate the hydrogens
What can NMR be used for?
- identify components / residues
What methods can be used for studying the activity of membrane channels / pores?
- use a radiolabelled solute or ion
- use ion selective fluorescent dyes
- some dyes change their fluorescent properties when binding to specific ions
What methods can be used for electrical measurements?
- voltage clamp
- patch clamp
- pure membrane
What is a quarts crystal microbalance used for?
- using quartz crystal microbalance to study the Mla pathway
What is the MLA pathway?
a system involved in maintaining the integrity of the outer membrane
