introduction to biospectroscopy - physics ...dutcher/download/phys...classifications of spectroscopy...
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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
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=
Heme (Porphyrin)-Based Pigments: Chlorophylls,
Cytochromes, and Globins
ChlA
CytCmyoglobin
From CooperFrom Alberts 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 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)
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
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)