Membrane Organisation and Energy Transfer Flashcards
1
Q
How does energy get to the LH1-RC complex?
A
- once energy cycles around B850 in LH2 it can hop to adjacent LH2s
- over time energy migrates to the LH1-RC complexes
2
Q
Purple Bacteria
LH1-RC Protein
LH1
A
- made up of 64 polypeptides, 80 transmembrane helices and 128 pigments
- 2 reaction centres wrapped in a broken figure of eight/S-shaped LH1
- around each RC in each half 28 B875 Chls for overlapping rings so 56 around each RC
- these B875s are sandwihed between inner and outer transmembrane polypeptides
- one carotnoid per BChl (so 56 per half) which have a spectral light harvesting role
- the S-shaped LH1 absorbs photons directly and accepts them from LH2
- rate of FRET in LH1 is typically faster than the rate of photochemistry in 1RC
- so if one RC is occupied an exciton can still access the second
3
Q
Purple Bacteria
LH1-RC Protein
RC
A
- remaining pigments are housed in the protein subunits that make up the RC
- energy has to be transferred to specialised Chls. the so called ‘special pair’
- an initial electron transfer process from the special pair creates a charge separated state or ‘ion pair’
- this photochemical reaction is the first point where electromagnetic energy has become chemical energy
4
Q
Path of Energy in Bacterial Photosynthesis
A
- excited energy is transferred energetically downhill by internal conversion within the LH2 B800 (10^-15 s)
- then from LH2 B800 to LH2 B850 then to LH1 B875 by FRET(10^-12 s)
- it’s then slightly uphill from LH1 B875 to Rc BChls but at ambient temperatures this energy difference is insignificant
- thermal energy at 298K is enough to make the jump
5
Q
Distance Between Chlorophylls
A
- for pairs of Chls and Chl-Car pairs Ro=3-8nm
- nearest neighbour distance between Chls within LH proteins ~0.5-2nm
- we may predict distance between proteins should be small, if this is the case energy transfer will proceed with high efficiency
6
Q
Protein Structure of the Reaction Centre
A
- L & M subunits integrated into membrane with helical portion of H subunit
- L & M contain 5 transmembrane alpha helices each
- each alpha helix ~4nm long so sufficient to transverse the membrane
- much of subunit H is in contact with L & M at the cytoplasmic side for stailisation
- H subunit caps the structure on the cytoplasmic side
7
Q
Reaction Centre
Arrangement of Cofactors
A
- symmetrically organised in 2 branches
- electrons transfer along the left branch only
8
Q
Reaction Centre
Primary Photochemical Reaction
A
- electron with in the special pair, PlPm becomes excited (->PlPm*) and is expelled
- electron very rapidly transferred via accessory BChl_L to BPh_L forming ‘primary ion-pair state’ (PlPm+ + BPh_L-)
- this takes <5ps
9
Q
Reaction Centre
Secondary Charge Transfers
A
- reduced BPh_L donates electron to adjacent Qa -> Qa- (200ps)
- Qa passes electron to nearby Qb (200μs) => Qb-
- this is electron tunnelling in biology
- cycle is repeate, the Qb can be used productively and electron from PlPm must be replaced
10
Q
Non-Radiative Relaxation Process
A
Dred + Aox – ket –> Dox + Ared
11
Q
Fermi’s Golden Rule
A
kif = 2π/ħ |Vif|² δ(Ei-Ef)
12
Q
Electron Transfer Rate from Fermi’s Golden Rule
A
ket = 2π/ħ |V~|² FC
- where V~ is the electron coupling between initial and final states, depends on the distance between donor and acceptor and relative orientations
- FC is the Franck-Cordon factor, viberational overlap integral (includes effect of temperature)
13
Q
Coupling
A
|V~|²(r) = Vo~² e^(-βr)
14
Q
Electron Transfer Rate as a Function of Distance
A
ket(r) = 2π/ħ Vo~² e^(-βr) FC
ket(r) ∝ e^(-βr)
15
Q
Franck-Condon Factor
A
- reaction rate depends on free energy change of the reaction, ΔGo
- and reorganization energy, λ, the energy required to distort the geometry of the reactants into geometry of products without energy transfer taking place
- both these factors are incoorporated into the Franck-Condon factor
16
Q
Thermally Activated Behaviour
A
- for electron transfers where: -ΔGo < λ or -ΔGo > λ
- there is ‘thermally activated behaviour’
17
Q
Activationless Behaviour
A
- for electron transfers where:
- ΔGo = λ
- FC is maximised so ket is maximal (very fast electron transfer) and there is only weak temperature dependence
- can neglect the integral in the FC factor and just keep the fraction
- this is the case for the reaction centre
18
Q
Relationship Between Time and Distance for Electron Transfer
A
-there is a positive linear correlation between ln(time) and distance time = 1/ket -and ket ∝ V~² ∝ e^(-βr) => time ∝ e^(βr) ln(time) ∝ βr ∝ r
19
Q
Placement of Cofactors
A
- photosynthetic systems are highly constrained, charge separation has to occur well within the fluorescence lifetime of the BChls (50ps)
- initial electron transfer events in the RC have to be fast, from Marcus theory this can only be acheived by placing the primary electron acceptor close to the special pair
- PlPm to BCh_L is ~6Å
- BCh_L to BPh_L is ~5Å
- this gives an overall transfer time of ~4ps from PlPm* to BPh_L
- the 10Å distance between BPh and Qa is ~200ps, also quite fast
20
Q
Photosynthetic Membranes in Purple Bacteria
A
- purple bacteria compartmemtalise their photosynthetic membranes into organelles, ‘intracytoplasmic membranes’ (vesicles)
- this creates an expansive system of photo-active membranes increasing the surface area for light absorption
21
Q
Why is membrane organisation important for photosynthetic function?
A
- absorption requires an extensive network of 1000s of Chls within the membrane held in place by antenna proteins
- the network of LHPs and RC complexes need to be physically extensive on a nanoscale
- the number and membrane distribution of RC traps needs to be optimal for high quantum yield of charge separation
- number and distribution of cytochrome complexes is important to ensure migration of electron carriers is not hindered by crowding of protein complexes in the membrane near the membrane surface
22
Q
What are the challenges of observing the organisation of proteins within native membranes?
A
- detailed structural information on complexes requires them to be removed from the membrane
- knowledge of original location in the native membrane is required for understanding
- methods of preparation for electron microscopy can alter/destroy native membrane organisation
23
Q
Solution to the Challenges of Observing Protein Organisation in Native Membranes
A
- atomic force microscopy
- images single molecules in membranes under liquid at near-native temperature, pH and ionic strength
- this preserves native membrane organisation
24
Q
How does AFM work?
A
- works by touch, sensitive to any vibration
- canitlever tip in contact with sample with laser point reflected off of canitlever onto detector
- as it bends to sample topology the canitlever moves and so does the laser spot
- feedback electronics adjust the cantilever to bring the laser spot back to the centre of the detector
- this vertical adjustment thus traces the surface topology of the model
25
Q
AFM Dimensions and Resolution
A
- cantilever length ~200μm
- cantilever thickness ~0.8μm
- tip height 2.9μm
- tip radius 20nm
- in-situ topographs have vertical resolution of ~1Å, lateral resolution of ~10Å
26
Q
Limitations of AFM
A
- some photosynthetic membrane proteins are not normally resolved by AFM because:
- -those proteins are present in lower numbers so harder to find
- -extraction process can remove them
- -they don’t protrude above the membrane as much so are harder to ‘see’ with AFM
27
Q
First Application of High Resolution AFM on Photosynthetic Membranes
A
- light harvesting membrane fragments extracted from purple bacteria
- AFM allowed observation of these ‘soft’ samples under liquids at lateral resolution ~1nm
- this was the first time any biological membrane with multiple proteins was visualised in ‘native’ state
- observed organisation of 10s-100s of light harvesting proteins in these membranes
28
Q
What would be required for modelling of solar energy cature across large biological membranes? (rather than single proteins)
A
- high res. 3D structueres of indiviudal light harvesting proteins (from x-ray)
- knowledge of ratio of one protein to another (from biochem. & spectoscopy)
- knowledge of spatial distances of proteins (AFM)
- knowledge of membrane superstructure (from EM)
- theoretical framework for excitation energy transfer (Forster theory)
- optimisation / verification of theory by comparison to experimental data from fluorescence spectroscopy
- computational power to render and simulate such a large model
29
Q
How is a model of the entire membrane produced?
A
- in principle it’s possible to reconstruct a model of the original membrane using information from several membrane patches
- and by putting known structures for the complexes at positions in the membrane according to the AFM map
30
Q
First Attempt at Modelling an Intracytoplasmic Membrane Vesicle
Description
A
- AFM images used to identify pigment-protein complexes with a planar patch
- multiple planar patches are combined and mapped onto a sphere using established area-conserving algorithms for sensible molecular spacings
- the available structural information is used to generate an atomic level model of the intracytoplasmic membrane (ICM) vesicle structure in silico
31
Q
First Attempt at Modelling an Intracytoplasmic Membrane Vesicle
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A
