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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 [email protected]

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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

[email protected]

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

Complementarity: IR and Raman

If molecule is centrosymmetric, no overlap of IR and Raman

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

A B

DNA VCD of PO2- modes in B- to Z-form transition

Experimental Theoretical

Z

B B, A

Z

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

Interferograms for different light sources

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

T

Synchrotron Light Sources – the next big thing

Broad band, polarized

well-collimated and

very intense

Light beam output

Where e-beam turns

Brookhaven National

Light Source

(and fixed in space!)