optical spectroscopy - brc · optical spectroscopy (light-matter interactions) is understood by the...
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Optical SpectroscopyTools to Investigate the Molecular Organization of Protein Complexes
Petar LambrevInstitute of Plant Biology
October 11, 2017
„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001
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OUTLINE
The essence of optical spectroscopy Photosynthetic membrane protein complexes
• Light-harvesting complexes• Reaction centers
Basic theoretical aspects • Molecular excited states and optical transitions• Exciton interactions and energy transfer
Polarized light spectroscopy – CD, LD, ACD, FP Time-resolved spectroscopy
• Ultrafast transient absorption spectroscopy• Time-resolved fluorescence spectroscopy
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THE ESSENCE OF SPECTROSCOPY
1. Find an object of interest2. Send a beam of electromagnetic radiation to the object3. Observe the outcoming radiation4. Learn something about the object
protein
light
lightprotein
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THE ELECTROMAGNETIC SPECTRUM
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INTERACTIONS OF EM RADIATIONWITH MATTER
• UV/VIS spectroscopy probes electronic excited states –electronic spectroscopy
• IR spectroscopy probes molecular vibrations –vibrational spectroscopy
Absorption Emission Reflection
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PHOTOSYNTHETIC ANTENNA COMPLEXES
dinoflagellates
PCP
LH2
purple bacteria
phycobilisomes
cyanobacteria
Fucoxanthin-chlorophyll-proteins
(FCP)
diatoms
LHC
plants
chlorosomes
green sulfur bacteria
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PHOTOSYNTHETIC PIGMENTS
Chlorophylls and bacteriochlorophylls• major photosensitive pigments• heterocyclic macrocycle• planar ring of conjugated π-bonds• central Mg atom• different types depending on
substitutents• Chls absorb blue (430-470 nm) and
red (640-660 nm) light
Carotenoids• xanthophylls – O-containing
carotenoids• linear chain of conjugated C=C
bonds
Carotenoid (β-carotene)
Chlorophyll a
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PHOTOSYNTHETIC PIGMENT MOLECULES
• Primary photochemistry only takes place in reaction center pigments
• Majority of pigments do not perform photochemistry – they are part of light-harvesting antenna complexes
• LHAs deliver absorbed light energy to RC via excitation energy transfer
The Emerson & Arnold experiment:
At saturating intensities, one O2molecule is produced per 2400 Chls
• 8-12 quanta are used per O2
• at least 4 by each photosystem• several hundred molecules associated
with each photosystem reaction center
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THE FUNNEL CONCEPT
• Energy is preferentially transferred downhill – from higher-energy states to lower-energy ones
• Higher-energy-absorbing pigments are located in the peripheral LHAs
• Lower-energy-absorbing pigments are located closer to the RC core
• Ensuring fast directional transfer towards the RC
• According to the Boltzmann distribution, in the thermally equilibrated antenna lower-energy states have higher population
blue-absorbing
green-absorbing
orange-absorbing
red-absorbing
Ener
gy
RC
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ENERGY FUNNELS IN PHOTOSYNTHETICORGANISMS
Purple bacteria Cyanobacteria
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LIGHT-HARVESTING COMPLEXES IN PLANTS
The LHC superfamily• Integral membrane proteins• 3 or 4 transmembrane helices• 10-15 chlorophylls as main
pigments• 2-4 xanthophylls as accessory
pigments• Monomeric or oligomeric• The protein determines the
pigments’ optical properties • Dynamic regulation of the light
harvesting function
Light-harvesting complex II
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LIGHT-HARVESTING COMPLEXES IN PLANTS
Lhcb1 (forms trimers) Lhcb3 (monomeric)
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LHCII TRIMERS
• Chls in LHCII are roughly arranged in rings, optimizing light absorption from all directions and energy transfer
• The energies of the pigment sites vary due to the protein environment and excitonicinteractions
• This creates a wider absorption band – more efficient light-harvesting
• Lowest energy pigments located in the periphery of the complex transfer energy away.
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PHOTOSYSTEM II CORE
top side
D1 (PsbA)D2 (PsbD)Cyt b559 (PsbE/F)
CP43 (PsbB)CP47 (PsbC)WOC (PsbO, PsbU, PsbV)
Accessory subunits(PsbF-PsbZ)
20-30 subunits35 Chlorophyll a
2 Pheophytin
11 β-carotene
2 Plastoquinone
2 Heme Fe
1 Non-heme Fe
4 Mn, 3-4 Ca, 3 Cl, HCO3, 20+ lipids
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PHOTOSYSTEM II SUPERCOMPLEXES
C2S2 C2S2M2
Nield & Barber, BBA, 2012, 1757:353-361 Pagliano et al., BBA, 2013, 10.1016/j.bbabio.2013.11.004
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PSII REACTION CENTERELECTRON-TRANSPORT CHAIN
Müh & Zouni, Front. Biosci., 2011, 16:3072-3132
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PHOTOSYNTHETIC PROTEIN COMPLEXES –SUMMARY
Proteins act as a smart scaffold that• bind a large number of light-absorbing pigments• dynamically control the properties of the bound pigments
The same pigment molecules can have a different functiondepending on the protein environment• Light-harvesting (in antenna proteins)• Light-dissipation (in antenna proteins under high light)• Photochemical reaction (in reaction center proteins)
Miniscule changes in protein conformation can change the pigment functions
The photophysical and photochemical reactions are ultrafast1 fs = 0.000 000 000 000 001 s
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LIGHT AS A WAVE
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POLARIZATION
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MOLECULAR ENERGY
Etotal = Evibrational + Eelectronic
E
S0-0
0-10-20-3
S1-0
1-11-21-3
S0-X – electronic ground stateS1-X – electronic excited stateSX-0 – vibrational ground stateSX-1 – vibrational excited state
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ABSORPTION OF LIGHT
Absorption of UV/VIS light is a molecular transition between two electronic levels
hυ
S0-0
0-10-20-3
S1-0
1-11-21-3
+–Dipole moment
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TYPES OF TRANSITIONS
• π π* C=C • n π* C=O (very weak)• d d Fe, Cu, Mn, Co
Compound λ [nm] ε [M–1 cm–
1]
-NH-CO- (π π*) 190 7000
-NH-CO- (n π*) 210 100
Trp 280 5600
Tyr 274 1400
NADH 340 14400
Chl a 660 76000
β-carotene 500 140000
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TIME OF THE TRANSITION
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DECAY OF THE EXCITED STATE
Fluorescence Internal conversion Vibrational
relaxation Intersystem crossing Phosphorescence Delayed fluorescence
• Energy transfer• Electron transferPerrin-Jablonski diagram
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ABSORPTION AND EMISSION SPECTRA
• Kasha-Vavilov rule: The fluorescence emission is independent from excitation wavelength
• Kasha’s rule (rephrased):Fluorescence is emitted from the lowest electronic excited level
• Mirror-image rule• Franck-Condon factors• Exceptions to the mirror image
rule
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RATE CONSTANTS, LIFETIMES AND YIELDS
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INTERMOLECULAR INTERACTIONS
Ionic Ion-ion interactions
Ion-dipole interactions
Van der Waals
Dipole-dipole interactions
Dispersion interactions
Debye dispersion interactions
London dispersion interactions
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THE EXCITONIC DIMER
Ea
Ea−V
Ea+V
2V
E
υ0a υ0a+Vυ0a−V
Abso
rptio
nmolecule 2
molecule 1
dimer transition dipole moments
monomer
dimer
Monomer and dimer energies
Interaction energy(point-dipole approximation)
x
y
z
1
2–
+
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FÖRSTER ENERGY TRANSFER
+ → +
A* B A B*
S0
S1
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THEORETICAL ASPECTS - SUMMARY
Optical spectroscopy (light-matter interactions) is understood by the theory of quantum electrodynamics
Light is portrayed as an electromagnetic wave Matter is portrayed as a set of molecular quantum eigenstates The electromagnetic field is coupled to transitions between electronic and
vibrational eigenstates Absorption and fluorescence emission spectra reveal information about the
molecular states Excited states decay via radiative and non-radiative pathways The excited state reaction pathways are characterized by rate constants The fluorescence lifetimes and yields reveal information about the excited-
state dynamics. Dipole-dipole interactions between molecules create new, shared exciton states Dipole-dipole interactions are the basis of energy transfer
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WHAT IS “POLARIZED LIGHT SPECTROSCOPY”?
Absorption• Polarized absorption• Linear dichroism• Circular dichroism• Magnetic CD• Anisotropic CD
– Chlorosomes– Chloroplasts– Thylakoid membranes– LHCII
Fluorescence• Fluorescence polarization • Fluorescence anisotropy• Fluorescence-detected
LD/CD
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The difference in absorption of light polarized parallel and perpendicular to an orientation axis.
LD gives direct structural information, because it depends on the angle of the transition dipole moment.
θ - angle between the transition dipole moment and main symmetry axis.
0 45 90 135 180
-0.5
0.0
0.5
1.0
LD /
3A
Angle, °
LINEAR DICHROISM
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SAMPLE ORIENTATION (ALIGNMENT)
Orientation by gel squeezing
Magnetic orientation
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ORIENTATION OF MEMBRANES
Face-aligned orientation should preferentially excite transitions in the membrane plane
Edge-aligned orientation shows transitions primarily perpendicular to the membrane.
However the linear anisotropy generates LD and distorts the CD
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The difference in absorption of left- and right-handed circularly polarized light.
CIRCULAR DICHROISM
35
• Intrinsic CD – chiral molecules• Excitonic CD – dipole interactions between pigments• Psi-type CD – long-range interactions in ordered pigment ensembles
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THE ORIGIN OF INTRINSIC CD
Pure electric absorption
Pure magnetic absorption
(current loop)
Optical activity
mμ ImCDRosenfeld equation:
mμ electric dipole moment
magnetic dipole moment
Cantor C.R. & Schimmel P.R., Biophysical Chemistry, 1980, Freeman & Co.
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EXCITONIC CD
• The two exciton transitions iof the dimer and have CD of equal magnitude but opposite sign
• The CD of the dimer is the sum of the CD of the two transitions
• The CD is nonzero because of the exciton energy split
• The monomer molecules do not need to be chiral
• The CD strongly depends on the geometry of the dimer
ν
(+) CD
(-) CD
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MEASURING LD AND CD
Photoelastic modulator: polarization is controlled by phase shiftingCD/LD is measured as the amplitude of the modulated signal
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CD OF CHLOROPLAST MEMBRANES
• Long-range pigment-pigment interactions in the membrane macrostructure produce psi-type CD
• Psi-type CD is sensitive to the macrostructural organization
• Disruption of the long-range interactions reveals the excitonic CD of pigment-proteins
CD spectra of stacked and washed thylakoid membranes
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CD OF INTACT PLANT LEAVES
C2S2
C2S2M2
koCP24
WT
CD
Wavelength (nm)
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CD OF ISOLATED LHCII
The CD spectra detect oligomerization and changes in the molecular environment
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TIME-RESOLVED SPECTROSCOPY
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TIME-RESOLVED SPECTROSCOPY
Information about fast and ultrafast processes in the system – excited-state reactions, etc.
The system is perturbed by a very short laser pulse and the time evolution of the system’s properties after the pulse is followed
Kinetic profile: can resolve multiple short-lived intermediate reaction states, their spectral properties, transient concentrations, reaction rate constants, etc.
Temporal resolution down to 1 fs = 0.000 000 000 000 001 s Temporal resolution and spectral resolution are related by the
uncertainty principle (short pulses have broad spectral width)
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PUMP-PROBE TRANSIENT ABSORPTION
• ‘Pump’ pulse creates excited states (GSS1)
• A subsequent ‘probe’ pulse (S1Sn) measures the changes induced by the pump
• The temporal evolution is followed by scanning over the time between pump and probe
• Temporal resolution is only limited by the pulse duration
GS
S1
S2pu
mp
pum
p
prob
epr
obe
Differential absorption: ΔA(t) = A+pump – A–pump
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PUMP-PROBE TRANSIENT ABSORPTION
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TRANSIENT ABSORPTION OF PHOTOSYNTHETICANTENNA COMPLEXES
Types of transient absorption signals: Negative A due to loss (bleaching) of ground states Negative A due to emission from excited states Positive A due to absorption by excited states
46
Active LH1 complexes
Energy-dissipating LH1 complexes
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TIME-RESOLVED FLUORESCENCE
Fluorescence lifetime:• Absolute value• Independent on
concentration• Insensitive to artifacts• Multiple lifetimes:
– Heterogeneity– True dynamics
F
t
A
τf
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INFORMATION FROM LIFETIME MEASUREMENTS
Fluorophore environment Multiple conformations, conformational changes Multiple environments Interactions with neighbouring residues Solvent relaxation Fluorescence lifetime sensors (Ca2+, Mg2+) Resonance energy transfer
Lakowicz J.R. (2006) Springer
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TRF QUENCHING
TRF can distinguish between • dynamic quenching (collisional quenching) – lifetime decrease with quencher
concentration• static quenching (exciplex formation) – lifetime is unchanged, amplitude
decreases
TRF can distinguish different quenched populations
Lakowicz J.R. (2006) Springer
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DECAY-ASSOCIATED EMISSION SPECTRA
A* B*kAB = 5 ns-1
A B
0.5 ns-1 0.5 ns-1
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METHODOLOGY FOR TRF SPECTROSCOPY
Direct
Gating
Frequency-domain (CW)
Phase modulation
Time-domain (pulsed)
TCSPC Streak camera Upconversion
TCSPC is the most versatile and commonly used technique
Can resolve lifetimes from few ps to μs High dynamic range and signal-to-noise ratio
Now affordable and accessible to non-specialist users
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TIME-CORRELATED SINGLE-PHOTON COUNTING
CFD
ADC
Memory
Detector
Reference pulsesfrom light source
Histogram
threshold
zero cross
CFD
threshold
zero cross
TAC
stop
start
Range
Gain
Offset
AddressAMP
data+1
Adder
(time)Preamplifier
= control elementsSingle-photonpulses
Time-to-amplitude conversion:1. The laser pulse starts a clock 2. The detected fluorescence photon stops the clock3. The time between the Start and Stop signals is recorded
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4. After many single photon events a histogram of decay times is collected
5. This histogram is the fluorescence decay kinetics
Original Waveform
Detector
Period 1
Period 5Period 6Period 7Period 8Period 9Period 10
Period N
Period 2Period 3Period 4
Resultafter
Photons
TimeSignal:
many
(Distribution of photon probability)
TIME-CORRELATED SINGLE-PHOTON COUNTING
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OPTICAL SPECTROSCOPY - SUMMARY
Polarized light spectroscopy is a valuable tool to monitor the molecular structure of protein complexes• LD reveals the orientation of chromophores in the protein matrix• CD is sensitive to short-range interactions of chromophores in the
protein and long-range interactions in protein macroassemblies
Time-resolved spectroscopy reveals excited-state reaction dynamics• Transient absorption can measure ultrafast transitions between
excited states, even nonradiative ones• Time-resolved fluorescence measures directly emissive excited states
and excited-state lifetimes with unparalleled precision, sensitivity and dynamic range
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THANK YOU FOR YOUR ATTENTION!
This work is supported by the European Union,
co-financed by the European Social Fund, within the framework of
" Practice-oriented, student-friendly modernization of the biomedical education for
strengthening the international competitiveness of the rural Hungarian universities "
TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.