3 - Optical Spectroscopy

Question 1

Define circular dichroism spectra

Answer 1

Can be used to estimate protein secondary structure.

It does not give the residue-specific information that can be obtained by X-ray crystallography or NMR.

Briefly, circular dichroism is defined as the unequal absorption of left-handed and right-handed circularly polarized light.

Question 2

How does circular dichroism work?

Answer 2

When asymmetric molecules interact with light, they may absorb right and left handed circularly polarized light to different extents (hence the term circular dichroism) and also have different indices of refraction for the two waves.

The result is that the plane of the light wave is rotated and that the addition of the ER (clockwise sinusoidal wave) and EL (counterclockwise sinusoidal wave) vectors results in a vector that traces out an ellipse and the light is said to be elliptically polarized.

CD is reported either in units of ΔE, the difference in absorbance of ER and EL by an asymmetric molecule, or in degrees ellipticity, which is defined as the angle whose tangent is the ratio of the minor to the major axis of the ellipse

Question 3

Give the approximate wavelength for the following spectroscopy methods:

• NMR
• Rotational
• Vibrational
• UV/visible spec

Answer 3

• NMR: Radio
• Rotational: Microwave
• Vibrational: Infrared
• UV/visible spec: Visible/UV

Question 4

Give the mechanisms associated with measuring the following types of absorption spectra:

• UV/vis
• IR
• Microwave

Answer 4

• UV/vis: valence electrons moving up or down atomic/molecular orbitals
• IR: Oscillation of molecular dipoles
• Microwave: Rotation of molecular dipoles

Question 5

What do spectrophotometers measure?

Answer 5

The amount of transmitted light as a function of wavelength

Transmitted wavelength = (Transmitted light intensity)/(incident light intensity)

Question 6

With UV/vis spectrometry, what happens to the electrons when light is passed through them?

Answer 6

Electron goes from occupied molecular orbital (typically the highest occupied) to an unoccupied MO

Question 7

What gives intensity variation (ε) in UV/vis spectra?

What gives individual ‘bands?’

Answer 7

Compatibility of molecular configuration between states (eg. HOMO and LUMO)

Individual bands are for distinct allowed ΔE transitions

Question 8

What type of electrons can be probed using UV/vis spectroscopy? (3)

Answer 8

• Lone pairs on oxygen
• Lone pairs on sulfur (very weak)
• π bonds

Question 9

What are the chromophores in proteins?

Answer 9

These allow a protein to be probed with optical spectroscopy

• Peptide bonds
• Aromatic side chains
• Sulfur containing side chains

Question 10

Define a isosbestic point

Answer 10

A wavelength at which the absorption of light by a mixed solution remains constant as the equilibrium between the components in the solution changes.

Eg. as the pH changes, the absorbance at a specific wavelength does not change (though it will change at others)

Question 11

What is the advantage of UV/Vis spectroscopy over nearly every other type?

Answer 11

That it can be used in real time to observe changes from modulating the environment.

It can also be used to observe the changes in protein conformation due to solvent perturbation etc.

It can also be used to monitor a reaction (such as enzyme catalysis). This is useful when studying extracts from mutant/wild type organisms, as an enzyme product may show an absorbance at a specific wavelength, permitting its presence to be determined.

Question 12

What is the advantage of circular polarized light, rather than plane polarized light in spectroscopy?

Answer 12

Circular polarized light is more likely to get absorbed by a chromophore, and therefore optical activity is much more observable and apparent.

Question 13

The absorption by a molecule depends on the direction of polarization of light, the molecule is optically active. This leads to what? (2)

Answer 13

• Different indices of refraction for left and right
  circularly polarized light: nL and nR
• Different molar absorption coefficients for left
  and right circularly polarized light: εL and εR

Question 14

How can the alpha, beta and disordered content of secondary structure be calculated from a CD spectra?

Answer 14

The features of each secondary structure add together. This allows you to calculate the percent composition of each secondary structure.

Eg: myoglobin
68.3% alpha helix
4.7% beta sheet
27% random coil

Question 15

What are two common applications of CD spectra?

Answer 15

Deconvolution: since Δε behaviour is additive, you can measure CD spectrum and use ideal Δε parameters to infer protein 2° structure content

Perturbations: Can very quickly demonstrate changes to protein structure (folded state) as a function of conditions (eg. temperature, urea etc.)

Question 16

Recall the relative rates of processes within fluorescence spectroscopy. (5)

Answer 16

Excitation: very fast

Vibrational relaxation/internal conversion: very fast

Emissive return from excited signlet state to ground singlet state: usually fast

Intersystem crossing: usually slow

Nonradiative return to ground state: varies

Question 17

In fluorescence, what is the energy state that is being excited from?

Answer 17

The lowest energy vibrational state of the ground singlet state

Question 18

In fluorescence, what is the energy state being emitted from?

Answer 18

The lowest energy vibrational state of the first excited singlet state.

Question 19

What are the two types of fluorophores in proteins?

Answer 19

• Intrinsic: aromatic side chains

- Extrinsic: bound within protein cavity or modify the protein chemically

Question 20

What is emission spectroscopy?

Answer 20

Where the emission wavelength and/or intensity from fluorescent molecules are observed

Question 21

What is red/blue shift with emission spectroscopy?

Answer 21

The wavelength of emission shifts depending on the environment.

Larger dipole moment in excited state than ground state allows for more pronounced environmental stabilization.

For example, in a more polar environment, DNS experiences a red shift (emits longer wavelength)

Question 22

Which three amino acids exhibit intrinsic fluorescence from best to worst?

Answer 22

Best
 - Tryptophan 
 - Tyrosine 
 - Phenylalanine
Worst

Question 23

Define quenching

Answer 23

Quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching.

It is a nonradiative way (no emittance) to return to the ground state.

Question 24

What are the two main mechanisms of quenching?

Answer 24

• Collisional (dynamic) quenching: collisions with quencher molecules return fluorophore to ground state without emitting a photon
• Static quenching: quencher molecule binds to fluorophore and changes ground state (and excited state) electron configuration such that complex is non-fluorescent

Question 25

What can quenching tell us about a protein’s structure?

Answer 25

Quenching can be indicative of binding. Titration with possible ligands can be used to produce quenching (with the right ligand and a fluorophore protein!)

Question 26

What are the applications of FRET quenching?

Answer 26

To study protein folding. Ie. the donor and acceptor positions/proximity induced by folding.

It can also be used for protein-ligand interactions, but only works when protein and ligand are in close proximity.

Question 27

The tertiary structure is influenced by the local environment. Is it observable by BOTH CD and absorbance spectra?

Answer 27

Yes!

Question 28

Explain FRET quenching in one sentence.

Answer 28

Proximity based energy transfer between suitable fluorophores.

Question 29

12C and 16O cannot be used in NMR spectroscopy, why?

Answer 29

Because they have no spin (I = 0)

Question 30

Which types of nuclei are active and useful in solution with an I of 1/2 for NMR spectroscopy? (5)

Answer 30

• 1H
• 13C
• 15N
• 19F
• 31P

Question 31

Which nuclei are active but quadrupolar for NMR spectroscopy? (5)

Answer 31

• 2H (deuterium)
• 14N
• 33S
• 23Na
• 17O