IR-spectroscopy

TheoryApplications to

biomolecules and proteins

Infrared spectroscopy

• Transitions between vibrational levels in the ground state

Lower energies than VIS

Infrared spectroscopy• Transitions between vibrational levels in the

ground state• The intensity is related to the transition dipole

moment• Dichroism can be observed in oriented states• Polymers give new spectral features compared

to monomers due to coupling between adjacent transition dipole moments

The intensity of a vibrational absorbtion band depends on the size of the transition dipole moment..

ba = <b || a >

How do wave functions look like for the ground state and the vibrationally excited state? NOTE: we must take both nuclei and electrons into account for vibrational transitions, since the nuclei are vibrating!

vv ’ (re , rn )

Born-Oppenheimer-approximation

We can divide the total wave function into

for the contribution of the elektrons

N for the contribution of the nuclei

(re : elektron coordinates, rn : coordinates of the nuclei) .

Then we can write the wave function as

State v State v ’

(re , rn ) =

(re , rn )

N (rn )

and the transition as

(re , rn )

Nv (rn ) (re , rn )

Nv’ (rn )

What do the wave functions look like for the ground state and the excited vibrational state?

NOTE: we must take both nuclei and electrons into account for vibrational transitions!

Born-Oppenheimer-approximation: We can divide the total wave function into contributions from the electrons () and contributions from the nuclei (). r: elektron coordinates, R: nuclear coordinates). Then we can write the transition as

The intensity of a vibrational absorbtionsband depends on the size of the transition dipole moment..

ba = <b || a >

But if the nuclei vibrate, it is possible to show by using taylor expansion ( where q = 0 is the equilibrium position of the nuclei) that the expectation value becomes:

If the nuclei are at rest the transition dipole moment only consists of , and becomes 0 since Nv, Nv’ are ortogonal

The alteration in dipole moment when the nuclei alter position

* Nv *

Nv’ d

*

d

Nv * Nv’ d

* Nv *

Nv’ d

* [ re q]q=0 d

Nv * q Nv’ d

If the dipole moment is altered during the vibration, IR absorbtion will occur!

Classical analogy:

= qR , /R = q

+q -qR The larger the charges, the larger the dipole moment – and the larger the absorbtion!

If the dipole moment of the molecule is altered during vibration, IR absorbtion will occur:

Biom

olecularIR absorbtion

band

VWe can see alterations in molecular binding character.

Identification of synthesized substances (-OH, -COOH, -COH)

Characterisation of material (most absorb IR)

Covalent bonds: enzymatic transitions

Non-covalent bonds: hydrogen bonds

Isotope shifts (1H mot 2H)

Hydrophobic bonds / VdW interactions: membranes, lipids

Protein structures

Biomolecular interactions

What does the sample cell look like?

ATR (attenuated total reflection): A film of protein or membrane is placed onto the IRS crystal

Measurements in KBr pellet is easiest

In solution: physiological, but you will have H2 O-absorbtion

Choose window with care! Must be transparent (BF,CaF, ZnSe, NaCl osv)

Cuvett length ~6 M

Effects of water

IR FT-IRSweep

the wavelength

region using

one

wave

length

at a timeFourier

Transform -

technologies

A large number of experiments to cover entire wavelength range

Number of ’successful’ experiments are few

A cord of frequences

Each

experiment is successful!

• Low signal-to-noise

• Takes time – most ’experiments’ give no result

• we get results rapidly

• adding up several experiments gives high sensitivity

First experimental confirmation of Watson-Crick base pairing came from IR spectroscopy

A+U

AU

G+C

GC

1:1 – base-to-base binding: evidence from IR titrations

Linear polarized light is widely applied in IR, since we can orient our samples onto the sample cell:

AII and A

can be related to molecular orientation.

Linear dichroism in oriented DNA molecules

Which curve shows T

and which shows

TII to the helix axis?

Linear dichroism in oriented DNA molecules

Vibration in the base plane results in larger absorbance/reduced transmission for light polarized parallel to the bases (orthogonal to the helix axis) since it is then parallel with the transition dipole moment.

From the difference between T II and T

the angle between the base plane and the helix axis can be calculated.

T

:

T II :

Difference spectra for two Ca2+-release reactions from phosphorylated (thin line) and non-phosphorylated ATP-ase (thick line)

Barth et al., JBC 1997

Covalent bonds: intermediates and products in enzymatic reactions

Time-resolved spectra (0.3-0.4 ms difference) from different photo-induced intermediates of bacteriorhodopsin

Can we observe protein structure?

Tsuboi, J. Poly. Sci., 1962

Poly-g-benzyl-L-glutaminsyra Heldragen linje: ljus polariserat parallellt med fiberriktningen

Streckad linje: polariserat ljus ortogonalt mot fiberriktningen

How sensitive is IR to secondary structure?

-helix

Random coil

-sheet

Interactions between transition dipoles renders transitions to be allowed or forbidden

Antiparallel -sheet -helix

Miyazawa, J Chem Phys, 1960

D10 > D30 > D20

How is secondary structure evaluated?

-Evaluation using the second derivative

-Fourier self-deconvolution (spectral deconvolution)

Example of evaluation process

-sheet

irregularDifferent kinds of -sheet

Steg 1 Steg 2

Membrane proteins we can study both the protein …

Bundle Monomer

Dynamic / 310 helix Dynamic/310 -helix

-helix-sheet/turn

Random coil

… and its effects on the lipid

ATR dichroism spectra of multibilayers without…

… and with protein: an organization!

Sal-Man et al., Biochemistry 2002, Preassembly of membrane active peptides is an important factor in their selectivity toward target cells.

Denaturation

Nativt tendamistat: denaturation midpoint (Tm) at 82 ºC

Mutated tendamistat: Lower Tm, aggregation at higher temperatures

Which situation results in significant changes in difference spectra?