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Going beyond normal IR spectra – FT variations
Linear Dichroism - Reflection (IRRAS)(courtesy Hinds Instruments)
Variable angleof incidence
Surface can be metal (high reflect., often grazing incidence)or dielectric, or even air-water interface
For film on liquid use Langmuir-trough for sample--studies of peptides and proteins in membranes possible, orientation from polarization
ATR Polarization MeasurementsIR beam multiply reflects inside crystal -- penetrates surface
keeps polarizations: Eperp in surface, Epara partially out
E⎥⎪
Sample coated on crystal
Crystal-Typ: Ge, ZnSeHigh index for refl.
In fromFTIR
out
ATR-FTIR C-H stretch 1,2-sn-dimyistoyl-phophatidylcholine film
ATR-FTIR of mellittinabsorbed to lipid monolayer
Difference from Lipid can be analyzed to give relative orientation
____ parallel, plane incidence- - - perpendicular
(Axelson,Dluhy,et al,App.Spec.1995)
Linear dichroism of a protein in membrane
A⎥⎪ - A⊥
A⊥
A⎥⎪
I II III
ApoL-p-III-DMPC complex – Goormaghtigh and co-workers, ACS Symp. 2000
Lipid
Possible orientation of a helical peptide in a membrane
In this situation, helix dipole (and amide I) polarized parallel to surface– Goormaghtigh and co-workers, ACS Symp. 2000
IRRAS- Looking at the liquid surface, focus on interface
From R. Dluhy website U.GA
IRAS of (KL4) 4K on an aqueous phospholipidDPPC monolayer
Cai, Flach, Mendelsohn
Increase surface pressure and sheet component appearsHelical component less variation
Lipid-gramicidin at air-water interface-polarized IRRAS indicate orientation
lipid
gramacidin
both
difference
Amide I vs. angleIncrease πs
Special Raman Techniques
Nonlinear Raman Spectroscopy: with extremely strong laser pulses (e.g. ~109 V cm-1 from Q-switched Nd-YAG lasers), higher order terms in induced dipole moment become important:
2 31 12 6P E E Eα β γ= + + +L
E: strength of applied electric field (laser beam)
α: Polarizability
β, γ: first and second hyper-polarizabilities
These lead to a whole collection of specialized experiments of which we willmention only a few. Modest impact on bio-systems
Coherent Anti-Stokes Raman Scattering -CARS
For sample irradiated by two high-energy laser beams at ν1 and ν2 (ν1 > ν2) in co-linear direction, beams interact coherently to produce strong scattered light of frequency 2ν1 − ν2. When ν2 is tuned to resonance such that ν2=ν1-νM for Raman active mode of sample at νM, a strong beam at frequency 2ν1 −ν2 = 2ν1 − (ν1 − νM) = ν1 + νM is emitted. Effect is called CARS (4-wave mixing). CARS signal is coherent and emitted (as a beam) in one direction and can be observed without a monochromator. It is on the anti-Stokes side and thus avoids fluorescence. All modes that are Raman active and some inactive Raman and IR modes are active in CARS.
CARS
Resonance Raman Spectroscopy
• Unlike the previous examples, RR is a popular technique for application to biomolecules—offers selectivity
-Vibrations strongly coupled to the chromophore are enhanced in Raman scattering intensity-In protein this can be the active site (e.g. heme or retinal groups)-Allows study of dilute systems-Power input (heating) to the sample is particular problem—flow cells-If excitation in uv is used, can single out aromatic side chains or if ~200 nm can excite amide modes
S1 -Real excited state
Ν− vibration
So-groundstate
UV-Resonance Raman –Excitation Wavelength Dependence
Manoharan, J.Microbiol.Meth. 1990
Aromatic modeswith low frequency excitations
Amide modes with high frequency excitation
Select out chromophore
Resonance Raman
• Excitation wavelength in resonance with functional group
• Allows measurements of dilute solutions and studies of specific parts of the molecule
SpectralSpectral--Structure Correlations for AmidesStructure Correlations for Amides
1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0
β - S h e e t
R a m a n S h i f t / c m - 1
Ram
an In
tens
ity
0
. 5
1 . 0
0
. 5
α - H e l i x0
R a n d o m C o i l
1 . 0
2 . 0
1235
1386
1551
1654
1299
1545
1647
1267
1386 15
60
1665
P u r e S e c o n d a r y S t r u c t u r e R a m a n S p e c t r a2 0 6 . 5 n m E x c i t a t i o n
C h i e t a l . , B i o c h e m i s t r y 3 7 , 2 8 5 4 - 2 8 6 4 ( 1 9 9 8 ) .
T h e s e b a s i s s p e c t r a a r e u s e d t o d e t e r m i n e t h e s e c o n d a r y s t r u c t u r e c o n t e n t o f p r o t e i n s .
Slide courtesy of Prof. Asher
coil
helix
sheet
RR like Raman and IR depend on frequency correlation, RR has focus on chromophore
SERS – Surface Enhanced Raman Spectroscopy
• Raman intensities have been shown to greatly increase is the molecules interact with small particles (colloids, nanostructures) of Au, Ag, Cu.
• Enhancement varies with λexcite, size and shape of particle as well as metal and molecule
• Enhancement for those molecles close to particle, so binding can be a problem if it introduces a structure change
Surface enhancement
• SERS is a surface enhanced Raman technique, now roughly 25 years old, and just coming to a state or reliability and huge enhancements – even single molecule
• SEIR(AS) is the IR complement, known for some time as >10X enhancement, but it is also coming on as a more understood and bigger effect due to plasmon resonance
Protein-lipid bilayer assembly on Au-coated surfaceReflection at the surface samples the bound membrane
SEIRAS from enhanced field at surface
Anthraquinone (AQ) on KRS-5a) 375 ng/cm2 AQb) same with 14 mm Ag colloidc) 125 ng/cm2 AQ/14mm Agd) 50 ng/cm2 AQ/14mm Ag
SEIRS enhancement d
c
b
a
Protein Functionality probed by SEIR:
The difference spectra reveal changes of the secondary structure induced by rearrangement of the hydrogen-bonded network among the internal amino acid side-chains surrounding the heme chromophore.
Anal Bioanal Chem (2007) 388 47-54
Surface enhanced Infrared absorption:
Raman Microscopy
IR Microscope – Tool for Chemical Imaging
Setup for visual inspection IR Setup
Reflective Objective
FTIR as illuminator
FTIR
Detector
(Wetzel & LeVine, 1990)
Advanced Techniques: FTIR Imaging
100 µm
1
100 µm100 µm
2 3
Amyloid deposit identification with FTIR
Amyloid deposits in neuronal cells. Evidence of amyloidformation in neuronal cells infected with spirochetes
J. Miklossy, et al., Neurobiol. Aging
From: Infrared Tissue Imaging Applications Growing in BiomedicalResearch and Diagnosis –Sharon Williams, Spectroscopy 2006
In prion protein diseases, some cells exhibit misfolded prion proteins (right cell), as evidenced by the elevated β-sheet content, whereas others do not (left cell)
Q. Wang, A. Kretlow, D. Naumann, and L.M. Miller, Vib Spect.(2005).
Stained section (left) and spectral pseudo color map (right) of lymph node.
Lymph node with colon cancer metastatis.
M. Diem, M. Romeo, et al., Analyst. 129(10), 880–885. (1994–2004). M.J. Romeo and M. Diem, Vibrational Spectrosc. 38, 115–119. (2005).
Other IR techniques• Photoacoustic looks at heat (sound) given off after
absorption, sense surface and can depth profile• Polymer stretching can evaluate molecular
response (polarized absorption) to macroscopic stress
• Ultrafast techniques can probe sample on– Nano sec scale – T-jump and pump probe– Femtosec scale – coherent 2-D and pump-probe
methods, depend on anharmonicity and coupling of non-degenrate modes
– Typically done dispersive with laser excitation/probe
Kinetics with IR forProtein folding
Keiderling group meeting, 07-June-06
Time dependent data with FTIR
Stop-flow methods - msec limits so far
Continuous, micro-flow methods - < 100 µsec
Rapid scan FT-IR - msec
Multichannel laser Raman, faster - µsec
T-jump and Flash photolysis -nsec time scalesusing step scan methods
Most T-jump single ν with tunable IR laser for S/N, filtering and . .
Scheme of Stop Flow—initialize by rapid mixing
Spacer
Mixer
GasketCell Window
Cell WindowFront Plate
Cell nest
Luer Plug
To Cell
Rea
gent
Prot
ein
Cell and mixer blowout
Syringe drive system
Mix protein and perturbant rapidly to get new state, follow spectra
Backplate
Time restriction from flow between windows and size
Time Dependent variation after Stop-Flow insertion
Qi Xu - unpublished
Qi Xu -unpublished
Exponential shows rate for H/D exchange, actually a fast and a slow process, surface and interior amides
This remains an interesting way to categorize folds!
Refolding of Ribonuclease A by FTIRInverse T-jump: Refolding initiated by injecting
Ribo A stored in syringe at 80 °C into IR cell at 25 °C
Wavenumber (cm-1)
15501600165017001750
log
(Si/S
f)
-0.03
-0.02
-0.01
0.00
0.01
0.021660 cm-1
loss of random coil
1630 cm-1
gain of sheet
time (s)
0 5 10 15 20
Peak
Inte
nsity
-0.03
-0.02
-0.01
0.00
0.01
1632 cm-1 (sheet)k = 0.156 s-1
1660 cm-1 (random coil)k = 0.342 s-1
Sheet refolding 2x slower than loss of coil
One single beam spectrum (IF scan) is collected for each time point. Time resolution = 50 ms, but could be faster, if modify. IR resolution 8 cm-1 sufficient to separate increase in sheet, decrease in coil as folds.
Callender/Dyer fluorescence T-jump setup
Generic design for T-jump, similar for IR (except transmit to MCT)
Cavity doubled, lots cw power
180o back scatter geom.
H2 gives 1.9 µ for D2O, CH4 ~1.5 µ H2O
Apo-Mb thermal unfolding, amide I
Very modest spectral change highly helical native state
2nd derivative shows mostly helical contribution
Difference spectra show loss of helix (1648) gain at 1673 cm-1
Gilmanshin, et al. PNAS 1997
Kinetic IR response to T-jump (45-60 C) - apo Mb
Solvated helix (1632 cm-1) lost very fast, ~100 ns, as is 1664 (turns?)protected helices (1655 cm-1) slower. Laser pulse heat water in 10’s ns
Gilmanshin, et al. PNAS 1997
Character of Temperature jump--timing
D2O - - -Sample ……Difference:a-1655 cm-1
b-1644 cm-1
c-1637 cm-1
d-1632 cm-1
Fit to biexpon.<10 ns160+/-60 ns
Helix example
D2O
10nsPump ∆T at 2µm focus to 300µm, 110 µm path use split cell
fast (50MHz) MCT detector, avg. 9000 shots, 10 Hz
3.0x10-5 to 4.0x10-5 (OD)/°C.µm for 1700 and 1632 cm-1
T-jump calibrated by change of D2O absorption with temperature
Williams. . .Dyer, Biochemistry, 35, 691, 1996
suc-FS 21-peptide: Suc-AAAAA-(AAARA)3A-NH2(Suc - succinyl, A - alanine, and R -arginine).
Helix T-jump, setup data
Analysis--partial sigmoid fit Stern-Volmer
Dynamic spectra match difference spectra
Time relaxation ~160 ns 2mM, ∆T ~20 C, ∆t ~ 20 ns heating
Williams. . .Dyer, Biochemistry, 35, 691, 1996
Squares = single frequency results spectrum by putting together
Advanced techniques
Multidimensional (2D)TeraHertz (farIR)Modulation Spectra (VCD)
2D IR Coherence Spectra—like NMR COSY
Pump one mode, see effect on other mode through time evolutionModes must be anharmonic and best if resolved
Figure from Woutersen web site
OPA, optical parametric amplifier; MCT, HgCdTe. M. Zanni Lab—U. Wisc.
Experimental 2D IR setup fs laser
2D IR uses 3 fs pulses, so 2nd excited states are measured. After heterodyning the response signal with a local oscillator pulse, 2D data set is collected and a FT along two time axes gives the 2D IR spectrum. Because overtone and combination bands are measured, 2D IR spectra exhibit cross peaks between coupled vibrational modes.
M. Zanni, Univ. Wisconsin
Cross-peaks (indicated by arrows) reflect couplings between C=O groups. From the couplings and anisotropies, solution conformation of the ring-thread system is determined on a sub-ps time scale. Fluctuations in conformation are observed with time-delayed 2D-IR.
2D IR dynamic conformationIn a molecular complex
Probing the structure of a rotaxane with 2D infrared spectroscopy - PNAS 2005O.F. A. Larsen, P.Bodis, W. J. Buma, J. S. Hannam, D. A.Leigh, S. Woutersen