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