INTRODUCTION TO BIOSPECTROSCOPY - Physics ...dutcher/download/phys...Classifications of Spectroscopy...

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INTRODUCTION TO BIOSPECTROSCOPY PHYS*4560/7510

Transcript of INTRODUCTION TO BIOSPECTROSCOPY - Physics ...dutcher/download/phys...Classifications of Spectroscopy...

INTRODUCTION TO BIOSPECTROSCOPY

PHYS*4560/7510

Electromagnetic Spectrum

ΔE = hc/λ = hν; ν - frequency in Hz, λ - wavelength in nm; 1/λ - wavenumber in cm-1; h – Planck’s constant 6.63×10-34 J×s; c = 3×108 m/s

UV vis NIR

γ-rays, X-rays

Radio

NMR

MicroMax Planck

Heinrich Hertz

Wavenumber

Wavelength

Classifications of Spectroscopy

1. By the electromagnetic spectrum region (electronic, vibrational, rotational)

2. By the kind of interaction with light– Absorption (transmission)– Scattering– Reflection– Refraction– Emission (fluorescence)– Change of polarization (dichroism)

3. By the time-domain– Static– Time-resolved – various methods of triggering– Ultrafast

Classifications of Spectroscopy

4. By the spectral manipulation– Absolute

– Difference

– Derivative

5. By the mode of acquisition– Direct

– From attached reporters

– From external reporters

6. By the number of wavelengths– Single-wavelength

– Scanning

– Multichannel (CCDs, diode arrays, interferometers)

Major General Applications

• To determine concentrations (D=εcl=log(Io/It))• To determine compositions of mixtures• To identify unknown compounds (fingerprinting)• To characterize kinetics of reactions and conformational

changes• To describe environmental changes of various chemical

groups• To determine orientation of certain groups• To find out mobility of the molecules• To figure out which groups are active in the processes of

interest and thus to establish mechanisms of these processes

Spectroscopies Useful for Biophotonics

Prasad, Introduction to Biophotonics (2003)

Transmittance and Absorbance of Light

• Transmittance T = It/Io [%]• Absorbance D = -log(T)= log(Io/It) [OD units];

1 OD = 10% transmittance• Beer-Lambert law D = εcl; where c is

concentration of the pigment, l is path length through sample, and ε is called molar extinction (or absorption) coefficient [l/mole/cm] (for example, ε is around 50,000-60,000 for visual reception molecules)

• Absorption spectrum - a plot of absorbance versus wavelength

UV/Vis (Electronic) Spectroscopy

From Siebert

Absorption spectra

http://www.biophysik.uni-freiburg.de/

Vibronic Structure of Electronic

Absorption Spectra

From Drago

Franck-Condon principle

Morse potentials

Ground state

Excited state

Electronic Spectroscopy: Remider of Some Important Concepts

• Relaxation of the excited state: heat, fluorescence, intermolecular conversion, photochemistry, intersystem crossing

• Oscillator strength (integrated intensity) f = 4.315x10-9x

• Transition dipole moment and dipole strength

• Singlet and triplet states

• Exciton splitting

∫ ννε d)(

v00dAA

ψψµ Μ= ∫ 200 || AAD µ=

Processes Involved in Electronic Excitation

Prasad, Introduction to Biophotonics (2003)

Typical Subjects of Biospectroscopy: Photosynthetic Pigments and Visual Pigments

From Lodish et al

Heme (Porphyrin)-Based Pigments: Chlorophylls,

Cytochromes, and Globins

ChlA

CytCmyoglobin

From CooperFrom Alberts et al

Chromophores of Biological Photosensors

From Hellingwerf

GFP (Green Fluorescent Protein) – an Example of Internally Formed Chromophore

From Tsien

Action Spectra vs Absorption Spectra

From Luttenberg et al

UV/Vis Spectroscopy: Drawbacks and Advantages

• Drawbacks:1. Wide overlapping bands – bad for fingerprinting

(but some have vibronic structure)2. Majority of biomolecules do not absorb in the

visible (only chromoproteins)3. Scattering is a big problem

• Advantages:1. Relatively easy to measure2. Easy to quantify (D=εcl=log(Io/It))3. Aromatic compounds have distinct environment-

sensitive bands in the UV

UV/Vis Spectroscopy: Instrumentation

• Static spectrophotometers1. Single-beam

2. Double-beam (sample & reference cells)

• Single-wavelength time-resolved spectrometers (flash-photolysis)

• Multichannel spectrometers1. Photodiode arrays

2. CCDs

Applications of UV/Vis Spectroscopy

• To quantify content of nucleic acids and proteins

• To follow kinetics of enzymatic reactions

• To study kinetics and thermodynamics of the reactions of photochromic proteins (flash-photolysis)

• To look at the environmental changes of aromatic amino acids

• To study access of the buried groups and topology of the proteins (stopped-flow)

Static Absorption Spectroscopy:

Titrations

Absolute

Difference

Isosbestic point

From Balashov et al

• Isosbestic point as an indicator of a binary mixture• Bathochromic (red) and hypsochromic (blue) shifts

Regular Spectrophotometers

Titration of a chromophore very often provides an information on a protein itself due to protein-chromophore interactions

UV Spectra of Aromatic

Amino Acids

From Tinoco et al

Titrations in the UV: Tyrosines

From Miura et al

UV Spectra Can be Used to Analyze Protein Secondary Structure

From Miura et al

Not very practical! CD and IR are much better suited

Typical Characterization of BR by Time-resolved Spectroscopy in the Visible

410 nm - follows protonation state of the retinal Schiff base

570 nm - follows disappe-arance of the initial state

660 nm - follows reisome-rization of the retinal and deprotonation of the primary proton acceptor

457 nm - in presence of pH-sensitive dye, follows H+ release and uptake

MO

H+N, BR

Flash-photolysis or Single-wavelength spectrometers

Multichannel Analysis

• Global exponential analysis of sets of difference spectra• SVD analysis• Varying temperature (or pressure or pH)

From Gergely et al

Diode Arrays and CCDs

Results of the Analysis:1. Spectra of the

intermediates2. Pathways of their

interconversions 3. Kinetics of individual

transitions4. Barriers of the reactions

From Gergely et al

Time-resolved UV/Vis Spectroscopy: Possible Artifact Sources

• Scattering and refraction index changes• Measuring beam effects• Photobleaching• Excessive excitation rate• Rotational diffusion plus polarized excitation• Actinic pulse duration (double-photon

reactions)

Additional Techniques To Supplement Spectroscopy in the Visible

• Low-temperature (cryo)• Stopped-flow (rapid-mixing)• T-jump• Pressure chamber• Double-flash• Optoacoustic and Photothermal (LIOAS and

PBD)• Linear Dichroism (LD)• Circular Dichroism (CD)

Use of Polarized UV/Visible Light In Biophysics: Reminder of Major Concepts

• Plane and Circular Polarization of Light

• LD (linear dichroism) D = (A║-A┴)/(A║+A┴)

• Optical Rotation α = (nL-nR)/λ

• ORD (optical rotation dispersion) – molar rotation vs wavelength

• CD (circular dichroism) εL- εR

• Ellipticity θ = 3298 (εL- εR)

Using LD to Detect Chromophore Orientation Changes

anisotropy

Order parameter

From Heyn

Basics of Circular Dichroism (CD)

• measures difference in absorptionof right- and left-circularly polarizedlight (R and L)

• absorption or extinction coefficients εR(λ) and εL(λ) are different

Δε(λ) = εL(λ) - εR(λ)

R & L have sameamplitude

R & L have diffamplitudes

plane-polarized elliptically-polarized

1: positive CD spectrum(L absorbed more than R)

2: negative CD spectrum(R absorbed more than L)

3: due to achiral chromophore

From Kelly et al.

Basics of CD

• usually plot molar ellipticity [θ] which is proportional to Δε

[θ] = 100 θ/C L

θ: observed ellipticity (rad)C: sample concentration (mol/l)L: path length (cm)

solid line: α helixlong dashed line: anti-parallel β-sheetdotted line: type I β-turncross dashed line: extended 31 helixshort dashed line: irregular structure

far UV CD spectra

From Kelly et al.

• absorption below 240 nm dominated by peptide bond– weak broad n → π* transition around 220 nm– more intense π → π* transition around 190 nm

CD Spectrophotometer

LS: light source (Xe 150 W) L: lensM: mirror F: filterP: prism SH: shutterS: slit PM: photomultiplierCDM: photoelastic modulator

Jasco J-810

single beam spectrophotometer

sample

• photoelastic modulator rocks polarization between R & L at frequency of ~ 50 kHz• use lock-in detection to measure difference in absorption

From Kelly et al.

Using UV-CD or ORD to

Detect Protein

Secondary Structure

CD

ORD

From Tinoco et al

• in addition to circular dichroism, can also have optical rotarydispersion ORD

– ORD specified by difference between nR & nL

φ = 180 L (nL – nR)/λ

[φ] = 100 φ/C L

CD Spectra

CD spectra of the threeconformations of poly-lysine

From Prasad

From Kelly et al.

Using Time-Resolved CD to Follow Protein Folding and Denaturation (Unfolding)

refolding of leucine zipper peptide using stopped flow CD

A: 6 µM B: 26 µM

Using CD in the Visible to Detect Protein Interactions

WTD85Nmutant

From Jang et al

From Kataoka et al

Excitonic Interaction of the Chromophores Responsible for the Biphasic Shape??

CD of Lipid-Protein Bilayers

190 200 210 220 230 240

M. Shimizu et al. Biochem. Biophys. Res. Comm. (2003)

cytochrome b562

CD of Gramicidin

-2

0

2

4

6

200 210 220 230 240 250 260 270

CD (m

Deg)

Wavelength [nm]

Gramicidin

Waterhelical dimer

(HD)double helix

(DH)

CD of Lipid-Protein Bilayers

J.B. Fiche, T. Laredo, O. Tanchak, J. Lipkowski, J.R. Dutcher and R.Y. Yada, Langmuir (in press)

CD of Lipid-Protein Bilayers

200 220 240 260 280 300

-3

-2

-1

0

1

2

3

200 220 240 260 280 3000.000.020.040.060.080.100.120.140.160.18

Abos

prtio

n (a

.u.)

Wavelength (nm)

A

Ellip

ticity

(mDe

g)

Wavelength (nm)

1

2

200 220 240 260 280 300-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

200 220 240 260 280 3000.00

0.02

0.04

0.06

0.08

0.10

Abso

rptio

n

Wavelength (nm)

Ellip

ticity

(mDe

g)Wavelength (nm)

B

suspension of DMPC:GD vesicles1: HD; 2: DH

~15 bilayers of DMPC:GD

J.B. Fiche, T. Laredo, O. Tanchak, J. Lipkowski, J.R. Dutcher and R.Y. Yada, Langmuir (in press)

CD of Lipid-Protein Bilayers

190 200 210 220 230 240 250 260-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

V=OCP V=+200mV V=0mV V=-200mV V=-400mV V=-600mV

∆ ≈ 1nmEl

liptic

ity (m

Deg)

Wavelength (nm)

∆ ≈ 1nm

effect of applied potential

J.B. Fiche, T. Laredo, O. Tanchak, J. Lipkowski, J.R. Dutcher and R.Y. Yada, Langmuir (in press)

CD of Lipid-Protein Bilayers

-600 -400 -200 0 200-2.2

-2.0

-1.8

-1.6

-1.4

-1.2

Potential (mV) vs. Ag/AgCl

Ellip

ticity

(mDe

g) a

t 200

nm m

easu

red

by C

D

50

60

70

80

90

100

110

120

130Film

thickness (nm)

measured by ellipsom

etry

Oriented films300mV>Δ ΦM-s >-150mV

Non-oriented film-150mV> Δ ΦM-s >-350mV

-600 -400 -200 0 200-2.2

-2.0

-1.8

-1.6

-1.4

-1.2

Potential (mV) vs. Ag/AgCl

Ellip

ticity

(mDe

g) a

t 200

nm m

easu

red

by C

D

50

60

70

80

90

100

110

120

130Film

thickness (nm)

measured by ellipsom

etry

Oriented films300mV>Δ ΦM-s >-150mV

Oriented films300mV>Δ ΦM-s >-150mV

Non-oriented film-150mV> Δ ΦM-s >-350mV

Non-oriented film-150mV> Δ ΦM-s >-350mV

J.B. Fiche, T. Laredo, O. Tanchak, J. Lipkowski, J.R. Dutcher and R.Y. Yada, Langmuir (in press)