Membrane Organisation and Energy Transfer

Question 1

How does energy get to the LH1-RC complex?

Answer 1

• once energy cycles around B850 in LH2 it can hop to adjacent LH2s
• over time energy migrates to the LH1-RC complexes

Question 2

Purple Bacteria
LH1-RC Protein
LH1

Answer 2

• 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

Question 3

Purple Bacteria
LH1-RC Protein
RC

Answer 3

• 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

Question 4

Path of Energy in Bacterial Photosynthesis

Answer 4

• 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

Question 5

Distance Between Chlorophylls

Answer 5

• 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

Question 6

Protein Structure of the Reaction Centre

Answer 6

• 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

Question 7

Reaction Centre

Arrangement of Cofactors

Answer 7

• symmetrically organised in 2 branches

- electrons transfer along the left branch only

Question 8

Reaction Centre

Primary Photochemical Reaction

Answer 8

• 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

Question 9

Reaction Centre

Secondary Charge Transfers

Answer 9

• 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

Question 10

Non-Radiative Relaxation Process

Answer 10

Dred + Aox – ket –> Dox + Ared

Question 11

Fermi’s Golden Rule

Answer 11

kif = 2π/ħ |Vif|² δ(Ei-Ef)

Question 12

Electron Transfer Rate from Fermi’s Golden Rule

Answer 12

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)

Question 13

Coupling

Answer 13

|V~|²(r) = Vo~² e^(-βr)

Question 14

Electron Transfer Rate as a Function of Distance

Answer 14

ket(r) = 2π/ħ Vo~² e^(-βr) FC

ket(r) ∝ e^(-βr)

Question 15

Franck-Condon Factor

Answer 15

• 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

Question 16

Thermally Activated Behaviour

Answer 16

• for electron transfers where: -ΔGo < λ or -ΔGo > λ

- there is ‘thermally activated behaviour’

Question 17

Activationless Behaviour

Answer 17

• 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

Question 18

Relationship Between Time and Distance for Electron Transfer

Answer 18

-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

Question 19

Placement of Cofactors

Answer 19

• 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

Question 20

Photosynthetic Membranes in Purple Bacteria

Answer 20

• 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

Question 21

Why is membrane organisation important for photosynthetic function?

Answer 21

• 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

Question 22

What are the challenges of observing the organisation of proteins within native membranes?

Answer 22

• 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

Question 23

Solution to the Challenges of Observing Protein Organisation in Native Membranes

Answer 23

• atomic force microscopy
• images single molecules in membranes under liquid at near-native temperature, pH and ionic strength
• this preserves native membrane organisation

Question 24

How does AFM work?

Answer 24

• 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

Question 25

AFM Dimensions and Resolution

Answer 25

• 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

Question 26

Limitations of AFM

Answer 26

• 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

Question 27

First Application of High Resolution AFM on Photosynthetic Membranes

Answer 27

• 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

Question 28

What would be required for modelling of solar energy cature across large biological membranes? (rather than single proteins)

Answer 28

• 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

Question 29

How is a model of the entire membrane produced?

Answer 29

• 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

Question 30

First Attempt at Modelling an Intracytoplasmic Membrane Vesicle
Description

Answer 30

• 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

Question 31

First Attempt at Modelling an Intracytoplasmic Membrane Vesicle
*****