biomolecular vibrational spectroscopy: part 1: principles of … · biomolecular vibrational...
TRANSCRIPT
T
BioMolecular Vibrational Spectroscopy:
Part 1: Principles of Infrared, Raman
Spectra and Techniques
Lectures for Warwick CD Workshop, Dec. 2011
Tim Keiderling
University of Illinois
at Chicago
T
Tentative Schedule — can vary with interests
Part I:
• Optical Spectroscopy (general)—low resolution, fast response
• Vibrational Theory
– Biologically relevant Vibrational Modes
– IR and Raman spectra - structure (qualitative)
• IR Instrumentation; FTIR principles
• Raman Instumentation
• Practical Demonstrations (lab? Break?) – background material
• Peptide methods—solution, solid
• Protein Sampling Techniques (aqueous), ATR
Part II:
• Application Examples
T
Structural Biology
• often need to know just the conformation
• structural determination of fold family may suffice,
generally not after atomic structure
• In BioTech processes one must monitor effect of
mutation and environmental changes
need to get this information rapidly and
in a cost effective manner
Measure all phases/types of samples
Look at fast time-scale events
Optical Spectroscopy is limited for determining
structure – lacks site specificity
but often fits important QUESTIONS
T
Near-IR
Electro-Magnetic Spectrum
Spectral
Regions
Wavenumber (cm-1)
Electron
Excitation
Electron
Transition
Molecular
Vibration
Molecular
Rotation
106 105 103 102 104 107 10 1
X-ray Ultraviolet Infrared Microwave
14,285 4,000 400 100
Mid-IR Far-IR
T
Vibrational Spectroscopy - Biological Applications
There are many purposes for adapting IR or Raman
vibrational spectroscopies to the biochemical,
biophysical and bioanalytical laboratory
• Prime role has been for determination of structure. We will
focus on secondary structure of peptides and proteins, but
there are more – especially DNA and lipids
• Also used for following processes, such as enzyme-substrate
interactions, protein folding, DNA unwinding
• More recently for quality control, in pharma and biotech
• New applications in imaging now developing, here sensitivity
and discrimination among all tissue/cell components are vital
T
Optical Spectroscopy - Processes Monitored
UV/ Fluorescence/ IR/ Raman/ Circular Dichroism
IR – move nuclei
low freq. & inten.
Raman –nuclei,
inelastic scatter
very low intensity
CD – circ. polarized
absorption, UV or IR
Raman: DE = hn0-hns
Infrared: DE = hnvib
= hnvib
Fluorescence
hn = Eex - Egrd
0
Absorption
hn = Egrd - Eex
Excited
State
(distorted
geometry)
Ground
State (equil.
geom.)
Q
n0 nS
molec. coord.
UV-vis absorp.
& Fluorescence. move e- (change
electronic state)
high freq., intense
Analytical Methods Diatomic Model
T
Essentially a probe technique sensing changes in the local environment of fluorophores
Optical Spectroscopy – Electronic,
Example Absorption and Fluorescence
Intrinsic fluorophores
eg. Trp, Tyr
Change with tertiary
structure, compactness (M
-1 c
m-1
)
What do you see?
(typical protein)
Amide absorption broad,
Intense, featureless, far UV
~200 nm and below
T
Optical Spectroscopy - IR Spectroscopy
Protein and polypeptide secondary structural obtained from
vibrational modes of amide (peptide bond) groups
Amide I
(1700-1600 cm-1)
Amide II
(1580-1480 cm-1)
Amide III
(1300-1230 cm-1)
Aside: Raman is similar, but different
amide I, little amide II, intense amide III
What do you see? – LOTS!
D
x 1
05
-4
-2
0
2
4
2000 1800 1600 1400 1200 1000
Wavenumbers (cm-1)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Absorb
ance I
II
III
800 1000 1200 1400 1600
0
1683ROA
1240
1426
1462
15541299 1342
1641
1665
2.6 x 105
IR -
IL
c) hen lysozyme
6.3 x 108
IR +
IL
0
1220
13451241
1658
16771295 1316
wavenumber / cm-1
4.7 x 105
ROA
IR -
IL
2.5 x 109
b) jack bean concanavalin A
IR +
IL
0
935
1640
166513001340
4.3 x 105
ROA
0
9.0 x 108
a) human serum albumin
IR -
IL
IR +
IL
800 1000 1200 1400 1600
0
1683ROA
1240
1426
1462
15541299 1342
1641
1665
2.6 x 105
IR -
IL
c) hen lysozyme
6.3 x 108
IR +
IL
0
1220
13451241
1658
16771295 1316
wavenumber / cm-1
4.7 x 105
ROA
IR -
IL
2.5 x 109
b) jack bean concanavalin A
IR +
IL
0
935
1640
166513001340
4.3 x 105
ROA
0
9.0 x 108
a) human serum albumin
IR -
IL
IR +
IL
800 1000 1200 1400 1600
0
1683ROA
1240
1426
1462
15541299 1342
1641
1665
2.6 x 105
IR -
IL
c) hen lysozyme
6.3 x 108
IR +
IL
0
1220
13451241
1658
16771295 1316
wavenumber / cm-1
4.7 x 105
ROA
IR -
IL
2.5 x 109
b) jack bean concanavalin A
IR +
IL
0
935
1640
166513001340
4.3 x 105
ROA
0
9.0 x 108
a) human serum albumin
IR -
IL
IR +
IL
Goal—try to give this meaning
T
Spectroscopic Process (covered)
• Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamically
changed when molecule is exposed to light
• In a spectroscopic experiment, light is used to probe a
sample. What we seek to understand is:
– the RATE at which the molecule responds to this perturbation
(this is response or spectral intensity – probability of transition)
– why only certain wavelengths cause changes (this is spectrum,
the wavelength dependence of the response – energy levels)
– the process by which the molecule alters the radiation that
emerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it
T
Spectroscopic Process (covered)
• Molecules contain distribution of charges (electrons and
nuclei, charges from protons) which is dynamically
changed when molecule is exposed to light
• In a spectroscopic experiment, light is used to probe a
sample. What we seek to understand is:
– the RATE at which the molecule responds to this perturbation
(this is response or spectral intensity – probability of transition)
– why only certain wavelengths cause changes (this is spectrum,
the wavelength dependence of the response – energy levels)
– the process by which the molecule alters the radiation that
emerges from the sample (absorption, scattering, fluorescence,
photochemistry, etc.) so we can detect it
T
Quantum mechanical picture
Full Hamiltonian describes electron and nuclear motion
H = - Sab [2/2Maa2 - 2/2mei
2 - Zae2/ria + e2/rij + ZaZbe
2/Rab ]
i.e. n-KE e-KE n-e attr. e-e repul. n-n repul
• Born-Oppenheimer approx. separate electron-nuclear w/f
y (r,R) = cu (R) fel (r,R) -- product fct. solves sum H
• Electronic Schrödinger Equation – issue for CD (done prev.)
Hel fel (r,R) = Uel (R) fe (r,R) – electron sol’n – nucl. pot.
Vn(R) = Sab [Uel(R) + ZaZbe2/Rab] – nuclear potential energy
• Nuclear Schrödinger Equation
Hn cu(R) = -[Sa (ħ2/2Ma) a2 + Vn (R)] cu(R) = Eu cu(R)
T
Solving Vibrational QM
• Nuclear Hamiltonian is 3N dim. – N atom, move x,y,z
– Simplify Remove (a) Translation (b) Rotation
– Result: (3N – 6) internal coordinates vibration
• Harmonic Approximation – Taylor series expansion:
V(R) = V(Re) + Sab V/RaRe(Ra-Re) +
½ Sab 2V/RaRbRe(Ra – Re)(Rb – Re) + …
– 3rd term –non-zero / non-const. - harmonic – ½ kx2
– Ra, Rb mixed Solution “Normal coordinates”
Qi = Sjcij qj H = -Si [2/2 2/Qi2+½ kQiQi
2] = Si hi (Qi)
hi ci(Qi) = Ei ci(Qi) Ej = (uj + ½) hnj solve as if independent
Diatomic: n = (1/2p) √k/m k – force const. m = MAMB/(MA + MB)
T
Harmonic Oscillator
Model for vibrational spectroscopy
re
r
e
r q
v = 1
v = 2
v = 3
v = 4
v = 0
hn 1
2 hn
3
2 hn
5
2 hn
7
2 hn
9
2 hn
E
re
Ev = (v+½)hn
Dv = 1
DE = hn
n = (1/2p)(k/m)½
(virtual
state)
Raman
IR
T
Spectral Regions and Transitions
• Infrared radiation induces stretching of
bonds, and deformation of bond angles –
• Couples like motions into molecular mode
• (ignore rotations for biomolecules in solution)
symmetrical
stretch
H-O-H
asymmetrical
stretch
H-O-H
symmetrical
deformation
(H-O-H bend)
T
Characteristic vibrations and structure
• heavier molecules bigger m - lower frequency
• H2 ~4000 cm-1 C–H ~2900 cm-1 C–D ~2100 cm-1
• HF ~4141 cm-1 HCl ~2988 cm-1
• F2 892 cm-1 Cl2 564 cm-1 I–I ~214 cm-1
• stronger bonds – higher k - higher frequency
• CC ~2200 cm-1 C=C ~1600 cm-1 C–C ~1000 cm-1
• O=O 1555 cm-1 N O 1876 cm-1 NN 2358 cm-1
• frequency depends mass + bond strength
T
Frequency structure, small and large molec.
Same for vibrational modes of amide (peptide bond) groups
Amide I
(1700-1600 cm-1)
Amide II
(1580-1480 cm-1)
Amide III
(1300-1230 cm-1) I II
a
b
rc
For polymer -- repeated structural elements have overlap/coupled spectra
T
Vibrational Transition Selection Rules
Harmonic oscillator: only one quantum can change
D vi = ± 1, D vj = 0; i j .
These are fundamental vibrations
Anharmonicity permits overtones and combinations
Normally transitions will be seen from only vi = 0, since most excited
states have little population.
Population, ni, is determined by thermal equilibrium, from the Boltzman
relationship:
ni = n0 exp[-(Ei-E0)/kT],
where T is the temperature (ºK) – (note: kT at room temp ~200 cm-1)
T ( r - re )/re
E/De
DE01 = hnanh--fundamental
D0—dissociation energy
Anharmonic Transitions
Real molecules are anharmonic to some degree so other transitions do
occur but are weak. These are termed overtones (D vi = ± 2,± 3, . .) or
combination bands (D vi = ± 1, D vj = ± 1, . .). [Diatomic model]
DE02 = 2hnanhrm - overtone
T
Vibrational Selection Rules • Interaction of light with matter can be described as the
induction of dipoles, mind , by the light electric field, E:
mind = a . E where a is the polarizability
• IR absorption strength is proportional to
~ |<Yf |m| Yi>|2, transition moment between Yi Yf
• To be observed in the IR, the molecule must change its electric dipole moment, µ , in the transition—leads to selection rules
dµ / dQi 0 relatively easy, ex. C=O str. intense
• Raman intensity is related to the polarizability,
I ~ <Yb |a| Ya>2, where da / dQi 0 for Raman trans.
T
Peak Heights
• Beer-Lambert Law:
• A = lc
– A = Absorbance
– = Absorptivity
– l = Pathlength
– c = Concentration
An overlay of 5 spectra of Isopropanol (IPA) in water. IPA Conc.
varies from 70% to 9%. Note how the absorbance changes with
concentration.
• The size (intensity) of absorbance bands depend upon molecular
concentration and sample thickness (pathlength)
• The Absorptivity () is a measure of a molecule’s absorbance at a given
wavenumber normalized to correct for concentration and pathlength – but as
shown can be concentration dependent if molecules interact
T
Peak Widths
• Peak Width is Molecule Dependent
• Strong Molecular Interactions = Broad Bands
• Weak Molecular Interactions = Narrow Bands
Water Water
Benzene
T
Level of structure
determination needed
depends on the
problem
Atomic resolution Ca chain
Secondary structure Segment fold (tertiary) 23
Structural
Biology
T
Chain conformation depends on f, y angles
Far UV absorbance broad, little fluorescence—coupling impact small
Detection requires method sensitive to amide coupling
If (f,y) repeat, they determine secondary structure
Polymer analysis Study the repeat units
T
Physical method of detection must sense
secondary structure — e.g. couple amides
IR/Raman— coupling comparable to band width, intensity
maximum is characteristic of structure – frequency basis
Circular dichroism --dipole and through-bond chiral coupling of
local modes (excitations) circularly polarized transitions,
DA = AL-AR - Develops characteristic band shapes (intensity)
Theoretically try to understand spectra/structure relation
IR ~ D=m.m~|dm/dQ|2 (Raman ~ |da/dQ|2)
ECD, VCD ~ R = Im(m.m)
Computable with ab initio QM techniques, ECD needs excited states
IR & VCD relatively easy, Raman more basis set sensitive
Major activity,
for analysis! }
T
Characteristic Amide Vibrations
I - Most useful;IR intense, less interference (by solvent, other modes,etc)
Less mix (with other modes)
II - IR intense
III - Raman Intense
A – often obscured
by solvent
IV – VII – difficult
to detect, discriminate
~3300 cm-1
~1650 cm-1
1500-50 cm-1
1300-1250 cm-1
700 cm-1
mix
T
Wavenumbers (cm-1
)
1450150015501600165017001750
Ab
so
rban
ce
0
1
2
3 helix
b-structure
randomcoil
IR absorbance spectra of selected model peptidesModel polypeptide IR spectra -- Amide I and II
Differentiation of conformations mostly due to coupling of amides
not to H-bonds or other factors, although they contribute
Helix—small frequency
dispersion, central ones
most intense, amide I,
higher ones for amide II
Sheet—large frequency
dispersion, characteristic
split amide I, broad amide II
Coil—less well-defined
broad amide I and II
I II
Frequency based
T
Temperature dependent IR
spectra of the helical peptide
Temperature dependence of
amide I’ frequency
IR frequency shift shows a sigmoidal curve and
spectra have an isobestic point for thermal unfolding
However, frequency shift is ~1635 ~1645 cm-1 – solvated helix
Monitoring structural change - temperature
folded
unfolded
T
6 b b sheet
, 2 )
Tyr97
Tyr25
Tyr92
H1
H3 H2
Tyr76
Tyr115
Tyr73
• 124 amino acid residues, 1 domain, MW= 13.7 KDa
• 3 a-helices
• 6 b-strands in an AP b-sheet
• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)
Ribonuclease A
combined
uv-CD and
FTIR study
Simona Stelea,
Prot Sci 2001
Optical spectra senses dynamic equilibrium - unstructured systems 29
T
Wavelength (nm)
260 280 300 320
Ellip
ticity
(mde
g)
-16
-14
-12
-10
-8
-6
-4
-2
0
Near-UV CD
Wavenumber (cm-1)
1600162016401660168017001720
Abso
rban
ce
0.00
0.01
0.02
0.03
0.04
0.05
0.06
FTIR
Wavelength (nm)
190 200 210 220 230 240 250
Ellip
ticity
(mde
g)
-15
-10
-5
0
5
Far-UV CD
Temperature 10-70oC
FTIR—amide I
Loss of b-sheet
Ribonuclease A
Far-uv CD Loss of a-helix
Near –uv CD Loss of tertiary struct.
Spectral Change 30
T
C i1 (x
102 )
-8.0
-7.6
-7.2
-6.8
-6.4
C i2 (x
10)
-1.0
-0.5
0.0
0.5
1.0
FTIRC i1
-17
-15
-13
-11
-9
-7
-5
C i2
-15
-10
-5
0
5
10
Near-UV CD
Temperature (oC)
0 20 40 60 80 100
C i1
-13
-12
-11
-10
C i2
-30
-25
-20
-15
-10
-5
0
5
Far-UV CD
Ribonuclease A
PC/FA loadings
Temp. variation
FTIR (a,b)
Near-uv CD
(tertiary)
Far-uv CD
(a-helix)
Pre-transition evident in far-uv CD and FTIR, not near-uv CD
Temp.
31
T
Nucleic acid IR
Nucleic Acids – less variation —helicity all about the same
a) – monitor ribose conformation
b) – single / duplex / triplex / quad – H-bond link bases
T
Sugars – little done, spectra broad, some branch appl.
Lipids – monitor order – self assemble – polarization
Example is CH2 wag, but
also stretch and scissor
bend are characteristic
Self assemble to lipid
bilayer – membrane
Polarization can tell
orientation of lipid or
protein in membrane
Other biopolymers
T
Combining Techniques: Vibrational CD “CD” in the infrared region
Vibrational chirality Many transitions / Spectrally resolved / Local
Technology in place DA ~10-5 - limits S/N / Difficult < 700 cm-1
Same transitions as IR
same frequencies, same resolution
Band Shape from spatial relationships
neighboring amides in peptides/proteins
Relatively short length dependence
AAn oligomers VCD have DA/A ~ const with n
vibrational (Force Field) coupling plus dipole coupling
Development -- structure-spectra relationships
Small molecules – theory / Biomolecules -- empirical,
Recent—peptide VCD can be simulated theoretically
T
Wavenumber (cm-1)
1600165017001750
Absorb
an
ce
0.0
0.5
1.0
DA
x 1
05
-10
-5
0
5
10
VCD
IR
(a)
Wavenubmer (cm-1)
1600165017001750
Ab
so
rba
nce
0.0
0.5
1.0D
A x
10
5
-4
-2
0
2
IR
VCD
(b)
Poly Lysine in D2O – Amide I’–Secondary structure
VCD
High pH – helix High pH, heating – sheet Neutral pH - coil
Wavenumber (cm-1)
1600165017001750
Absorb
ance
0.0
0.5
1.0
DA
x 1
05
-15
-10
-5
0
5
IR
VCD
(c)
T
-1
VCD of DNA, vary A-T to G-C ratio
base deformations sym PO2- stretches
big variation little effect
All B-DNA forms
T
800 1000 1200 1400 1600
0
1683ROA
1240
1426
1462
15541299 1342
1641
1665
2.6 x 105
IR - IL
c) hen lysozyme
6.3 x 108
IR + IL
0
1220
13451241
1658
16771295 1316
wavenumber / cm-1
4.7 x 105
ROA
IR - IL
2.5 x 109
b) jack bean concanavalin A
IR + IL
0
935
1640
166513001340
4.3 x 105
ROA
0
9.0 x 108
a) human serum albumin
IR - IL
IR + IL
Protein RAMAN & ROA spectra
hSA
Con A
HEWL
I III
ROA sign
patterns
stable but
frequencies
shift.
Chirality
selects out
amide modes
but Raman
spectra
dominated by
aromatics
Barron data
T
IR & Raman Instrumentation - Outline
• Principles of infrared spectroscopy
• FT advantages
• Elements of FTIR spectrometer
• Acquisition of a spectrum
• Useful Terminology
• Mid-IR sampling techniques
– Transmission
– Solids
• Raman instrumentation comparison
• (Note—more on sampling variations later)
T
Dispersive spectrometers (old) measure transmission as a function
of frequency (wavelength) - sequentially--same as typical UV-vis
Interferometric spectrometers measure intensity as a function of
mirror position, all frequencies simultaneously--Multiplex advantage
Sample
radiation
source transmitted
radiation
Techniques of Infrared Spectroscopy
Infrared spectroscopy deals with absorption of radiation--detect attenuation of beam by sample at detector
Frequency
selector
detector
T Nicolet/Thermo drawings
Comparison of IR Methods –
Dispersive & Fourier Transform
But add to this now many laser-based technologies!
T
New specialized experiments still use dispersive IR
T/jump IR with
diode laser
Dispersive VCD for Bio Apps
2-D IR setup with 4-wave mixing
T
Major Fourier Transform Advantages
• Multiplex Advantage
– All spectral elements are measured at the same time,
simultaneous data aquisition. Felgett’s advantage.
• Throughput Advantage
– Circular aperture typically large area compared to dispersive
spectrometer slit for same resolution, increases throughput.
Jacquinot advantage
• Wavenumber Precision
– The wavenumber scale is locked to the frequency of an internal
He-Ne reference laser, +/- 0.1 cm-1. Conne’s advantage
T
Typical Elements of FT-IR
IR Source (with input collimator)
– Mid-IR: Silicon Carbide glowbar element, Tc > 1000oC; 200 - 5000 cm-1
– Near IR: Tungsten Quartz Halogen lamp, Tc > 2400oC; 2500 - 12000 cm-1
IR Detectors:
– DTGS: deuterated triglycine sulfate - pyroelectric bolometer (thermal)
• Slow response, broad wavenumber detection
– MCT: mercury cadmium telluride - photo conducting diode (quantum)
• must be cooled to liquid N2 temperatures (77 K)
• mirror velocity (scan speed) should be high (20Khz)
Sample Compartment
– IR beam focused (< 6 mm), permits measurement of small samples.
– Enclosed with space in compartment for sampling accessories
T
Interference - Moving Mirror Encodes Wavenumber
Source
Detector
Paths equal all
n in phase
Paths vary
interfere vary for
different n
T
Single, double or
triple monochromator
Detector:
PMT or
CCD for
multiplex
Filter
Polarizer
Lens
Sample
Laser – n0
Dispersive Raman - Single or Multi-channel
Eliminate the intense Rayleigh
scattered & reflected light
-use filter or double monochromator
–Typically 108 stronger than the
Raman light
•Disperse the light
onto a detector to
generate a
spectrum
Scattered Raman - ns