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

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

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

THE ELECTROMAGNETIC SPECTRUM

INTERACTIONS OF EM RADIATIONWITH MATTER

• UV/VIS spectroscopy probes electronic excited states –electronic spectroscopy

• IR spectroscopy probes molecular vibrations –vibrational spectroscopy

Absorption Emission Reflection

PHOTOSYNTHETIC ANTENNA COMPLEXES

dinoflagellates

PCP

LH2

purple bacteria

phycobilisomes

cyanobacteria

Fucoxanthin-chlorophyll-proteins

(FCP)

diatoms

LHC

plants

chlorosomes

green sulfur bacteria

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

7

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

8

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

9

ENERGY FUNNELS IN PHOTOSYNTHETICORGANISMS

Purple bacteria Cyanobacteria

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

LIGHT-HARVESTING COMPLEXES IN PLANTS

Lhcb1 (forms trimers) Lhcb3 (monomeric)

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.

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

PHOTOSYSTEM II SUPERCOMPLEXES

C2S2 C2S2M2

Nield & Barber, BBA, 2012, 1757:353-361 Pagliano et al., BBA, 2013, 10.1016/j.bbabio.2013.11.004

PSII REACTION CENTERELECTRON-TRANSPORT CHAIN

Müh & Zouni, Front. Biosci., 2011, 16:3072-3132

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

LIGHT AS A WAVE

POLARIZATION

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

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

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

TIME OF THE TRANSITION

DECAY OF THE EXCITED STATE

Fluorescence Internal conversion Vibrational

relaxation Intersystem crossing Phosphorescence Delayed fluorescence

• Energy transfer• Electron transferPerrin-Jablonski diagram

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

RATE CONSTANTS, LIFETIMES AND YIELDS

INTERMOLECULAR INTERACTIONS

Ionic Ion-ion interactions

Ion-dipole interactions

Van der Waals

Dipole-dipole interactions

Dispersion interactions

Debye dispersion interactions

London dispersion interactions

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–

+

FÖRSTER ENERGY TRANSFER

+ → +

A* B A B*

S0

S1

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

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

31

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

32

SAMPLE ORIENTATION (ALIGNMENT)

Orientation by gel squeezing

Magnetic orientation

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

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

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.

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

MEASURING LD AND CD

Photoelastic modulator: polarization is controlled by phase shiftingCD/LD is measured as the amplitude of the modulated signal

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

CD OF INTACT PLANT LEAVES

C2S2

C2S2M2

koCP24

WT

CD

Wavelength (nm)

CD OF ISOLATED LHCII

The CD spectra detect oligomerization and changes in the molecular environment

TIME-RESOLVED SPECTROSCOPY

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)

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

PUMP-PROBE TRANSIENT ABSORPTION

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

TIME-RESOLVED FLUORESCENCE

Fluorescence lifetime:• Absolute value• Independent on

concentration• Insensitive to artifacts• Multiple lifetimes:

– Heterogeneity– True dynamics

F

t

A

τf

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

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

DECAY-ASSOCIATED EMISSION SPECTRA

A* B*kAB = 5 ns-1

A B

0.5 ns-1 0.5 ns-1

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

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

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

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

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.