CD Spec

Question 1

What main thing does CD spec measure

Answer 1

The optical activity of molecules

Directly Measures the delta A and delta extinction coefficient (the diffence in how much or how strongly light is being absorbed)

Question 2

What type of transitions does CD use

Answer 2

Looks at electronic transitions (pi to pi star or n to pi star) in the UV range

Question 3

What are the two main regions we conduct CD experiments and why

Answer 3

Far UV (178-260 nm): absorabnce from the backbone amides gives psecondary structure percentage (like how hemoglobin is 70% alpha helix)

Near UV (260-320): absorbance form aromatic side chains (trp tyr) gives changes in tertiary structure due to mutations denaturation or ligand binding

Question 4

What are non bonding orbitals

Answer 4

Orbitals that don’t contribute to bonding or antibonding

Question 5

What are bonding orbitals

Answer 5

Either sigma or pi orbitals

Sigma: head on head overlap of two atomic orbitals (along inter nuclear axis)

Pi: sideways overlap of two P atomic orbitals (above and below nuclear plane)

Bonding orbitals are formed from constructive interference of the atomic orbitals

Question 6

What are antibonding orbitals

Answer 6

Sigma star and pi star, same as the bonding orbitals but due to destructive interference instead of contraictive

Question 7

Bonding antibonding and non bonding are what type of orbitals

Answer 7

Molecular orbitals and they interact through atomic orbitals

Question 8

Between all the movlular orbital what is the energy difference

Answer 8

Bonding sigma then pi :lowest

Non binding : medium

Antibonding pi then sigma : highest

Question 9

Describes the strength of the n to pi star transition in regular absorbance and in CD

Answer 9

In regular absorbance :

The n to pi star transitions are very much weaker (e < 100 M-1cm) than the strong pi to pi star (>1000M-1cm-1)

This means pi to pi star absrobs more light and n to pi star absorbs less

In CD absorbance:

The n to pi star transition ate important and have similar intensity/ delta e as pi to pi star

Question 10

Where do pi to pi star and n to pi star transition occur

So overall what experience transitions

Answer 10

n to pi star is in the amide backbone

Pi to pi star is in tyr or trp residues

Carboxyl, carbonyl and OH groups, not lone pairs

Question 11

At what wavlngth do pi to pi star and n to pi star absorbance transtions occur

Answer 11

Pi to pi star : needs higher energy to transition/absorb , lower wavelength (192 nm)

n to pi star : needs lower energy to transition/absorb, higher wavelength (220 nm)

Question 12

Whag is the wavlngth scale of cd

Of Fluor

Answer 12

Far UV: looking at amides in backbone, pi to pi star and n to pi star (180-260), can also get the aromatics but most signal comes from amides

Near UV: looking at trp tyr phe pi to pi star and n to pi star (260-320), there’s also disulphides that contribute but negligible

Fluor is visible region: 400-700

Question 13

In understanding polarized light what are light waves

Answer 13

Light waves are a wave of an electric field (E) oscillating in one direction (up down) and the magnetic field (B) occsilating perpendicular to E (back forth)

Both E and B are perpendicular to the direction of propagation (forward)

Question 14

In understanding polarized light what is unpolarized light

Answer 14

From the continuously oscillating magnetic and electric fields, there is the resulting electric field vector (magnitude and direction of the electric field)

In unpolarized light, there are many different electric fields in different orientation, so the electric field vector oscillates in all random directions perpendicular to the direction of propogation

Question 15

In understanding polarized light what is polarized light

Answer 15

Only one electric field selected, so The electric field vector oscillates on a single fixed plane (in one direction)

Question 16

What is the transition dipole moment and how does it relate to CD

Answer 16

Mew knot: the direction and amount of electron density in a chromoohore (vector)

(dipole is disparity of change between on side and the other of the chromoohore, the mew knot is a vector that describes the direction and magnitude

In CD: If mew knot vector in the chromoohore aligns with the electric field vector (from the light source), more light is absorbed

Question 17

What is circularly polarized light

Difference in circular vs plane polarized light

Answer 17

Instead of oscillating up and down like in polarized light, the electric field vector oscillates in a circle around the direction of propagation

This is because of a 90 degrees phase shift of the E and B waves (causing them to not be aligned and vector to be in circular direction)

Circular: E vector has constant amplitude, rotating between axis

Plane polarized (linear): changing amplitude along constant axis

Question 18

In what direction can circularly polarized light be polarized

Answer 18

Can be CW or CCW

Left circular polarized light (LCPL) is CCW, right (RCPL) is CW : ONLY WHEN LOOKING DIRCTION OF PROPOGATION FACING YOUZ

if direction of propogation is facing away, it’s opp

In chiral molecules(amides trp and tyr with electronic transitions), the RCPL AND LCPL are absorbed in different amounts

Question 19

When is a wave elliptically polarized

Answer 19

If the phase difference wasn’t 90 degrees (1/4 cycle) or if the waves had diff amplitudes

Question 20

Describe the CD spectrometer

Answer 20

Called a spectropolarimeter

Light source : Xenon l

Circular polarizer: Rapidly alternates between making L or R circular polarized light (RLRLRL)

The L or R are absorbed differently by diff chromoohore so by alternating, you find the changes in in absorbtion of L and R, this gives changes in delta E

Very short cuvettte path length (0.1 mm)

Electronics: amplifies the signal

This gives delta e vs wavlngth spectra

Question 21

What is the output of a soectropolarimeter

Answer 21

If the AL (absorabce of LCPL) doesn’t equal AR you get delta A: delta A= AL-AR

Delta A is proportional to observed ellipticity (theta) and is the main output of CD (so cd doesn’t report the delta A but the ellipticity)

From observed ellipticity this you get MRE or molar ellipticity

Can also have delta e as an output

Question 22

How does CD spectra look diff than absorbance soectra

Why is this important

Answer 22

Abs: e vs wavlngth the peaks are always postive

Cd: delta e vs wavlngth, peaks can be positive negative or almost zero since looking at the change in delta e
In simple cases the wavlngth of CD peaks are the same as their abs peaks

In abs the e is big but in CD the delta e is small, this means CD is a sensitive technique that detects small changes in e

Question 23

What did early instruments use to measure CD and how

Answer 23

Intstead of CPL, ised plane polarized light,

the delta e was measured by ellipticity

incoming light: is plane polarized (theta magnitude of R and L equal) and has no ellipticity (theta = 0)

outgoing light: the uneven R and L absorption of the oscillating E vector by a chiral molecule leads to elliptically polarized light . The vector shifts to the by some dergrees which is theta

LOOK OVER AGAIN

Question 24

What are the two types of ellipticity you can get from CD

Answer 24

CD machine gives theta obs

Molar ellipticity: based on try MW of protien

units are deg*cm^2/dmol

= theta obs (in degrees) x MW of the protien x 100 / path length x concentration of protien

Used in near UV looking at tertiary structure

Mean residue ellipticity (MRE):

Units are deg*cm^2/dmol

using the average MW of the residues in the protien

= delta e x avg MW of residues (cm3 deg/mol)

More common for bigger protiens because get higher value if multiplying by MW rather than mean residue MW

Used in Far uv looking at secondary structure

Question 25

Example wuestion for finding MRE

Answer 25

Can do two ways: If don’t have delta e, find the mean residue MW , put into molar ellipticity question in place of protien MW Or if have delta e, multiply 3300 constant to get MRE Using molar ellipticity equation If given MW convert to g/mol: KDA: kda x 10^3 Da: just the da value Divide that by number of residues to get mean residue MW Convert millidegrees to degrees: millix10-3 Mg/mL = g/dm3 Delta e way: need delta e If want to look for delta e too: Delta e = mean residue Ellipticity/ conversion factor

Question 26

1L=1dm3 1mol=10dmol 1 dm3 =1000cm3

Answer 26

Okay

Question 27

In the CD spectra Whag can be on. The y axis

Answer 27

Delta e, molar ellipticity, MRE

Question 28

What is special about the CD spectra of a protien in the 180-260 region (far UV)

Answer 28

Each type of secondary structure (alpha helix beta sheet random coil) has a distinct shape and magnitude in the far UV region It’s the average of the secondary structural parts of the protien Signal is coming from the backbone amides

Question 29

Explain the characteristics of the CD soectra at 178-260nm (far UV)

Answer 29

Alpha helox: intense band at 192nm, delta e > 10 M-1cm-1 at 192nm, double dip around 208 and 222nm Beta sheet: single postive at 198nm, single negative 218nm, delta e 192 < 3, delta e 195 <10 Random coil: negative at 198nm, only one that’s postive at 218nm For mixed protiens: see intensity of beta but shape of alpha heloxe, double dip 200-230 nm region, 193-195 delta e<10 and 3

Question 30

Explain why there is a double dip in the alpha helix peak in far uv CD

Answer 30

Chromoohore coupling: the chiral chromoohores in the helix are coupled (close together) They each give opposite signs for pi to pi star transitions which gives two separate peaks (the large postive and the next negative) So it’s a single pi to pi star transition but shows two peaks of opp sign due to coupling So alpha helix peak is due to pi to put star postive, then negative then n to pi star negative (both negatives make the double dip)

Question 31

Give the hemoglobin, lysozyme, elastase example of far UV CD

Answer 31

Hemoglobin shows mainly alpha helix peak because mainly alpha helical Lysozyme is mixed so it shows some double dip, but not the big alpha helix peak at 192nm Elastase: random and unfolded, shows negative at 195 then goes to postive at higher wavlngth

Question 32

How can you distinguish between beta sheet and beta turn in CD spec

Answer 32

Same shaped, beta turn is shift to the longer wavelength (right)

Question 33

How can you quantitatively find the fraction of a certain secondary structure in CD

Answer 33

Use the equation where fraction of each =1 Helix, antiparallel, parallel beta sheet, beta turns, other (random coil usually) All of these elements add up to the structure of the protien as a whole

Question 34

What is a basis soectra

Answer 34

A far UV CD soectra of a protien that you know for sure has a single secondary structure Then Compare your proteins cd spectra to the basis spectra to find the proportion of that type of secoarndy structure in your protien

Question 35

What are the problems with using basis soectra

Answer 35

1. How do we know which basis spectra to use: one from polypeptides? One from other protiens with a known secondary structure? 2. We assume that only amides contribute to the cd in the far uv (178-260), but higher energy transitions from aromatics residues (trp tyr phe) will contribute (we assume they aren’t a large factor) The basis soectra from protiens can account For this. if you get the basis spectra of protiens that have a similar number of aromatic amino acids and they have a single well defined secondary structure you can use those to avoid this effect.

Question 36

How does the CD spectra in the near uv (260-320) work How are changes in tertiary structure happening in this region

Answer 36

the aromatic side chains are not chiral molecules like amides so normally don’t have cd signal But Because of the disymmetric regions in the protien, the CD signal get induced in the aromatic side chains which shows up in near uv This cd signal is sensitive to tertiary structure BUT CANNOT DETERMINE IT Change happen by: Ligand binding Interaction of subunits Proteolytic cleavage Denaturation Effect of Mutations on protien domains

Question 37

Explain disymmetry

Answer 37

Mocleule has no planes or points of symmetry, may or may not have an axis of symmetry Basically chiral molecules (no symmetry, can’t superimpose on its mirror image) When put an aromatic side chain in this disymmetric environment, this induces it to give RPCL or LCPL resulting in the near UV spectra

Question 38

Practice problem 1: is tyrosine hydoxylase highly alpha helical

Answer 38

Step 1: start with far UV (178-260) secondary structure: Gave WT NTD and CTD WT and CTD: has MRE >10 at 195 nm (aloha helix) has Double dip at 208 and 222 (alpha helix) NTD: weak signal all across, suggests unstructured random coil, small postive at start suggest maybe beta sheet Since WT similar to CTD and both show mainly alpha helical, protien mainly alpha helical with CTD contributing most Step 2: move to with near UV (260-310) tertiary structure: CTD AND WT: similar, small change in tertiary structure around trp or tyr in protien in the CTD compared to WT NTD: little to no signal, meaning no dissymatery induced in the NTD, meaning it’s mainly random coil (since disymmetry doesn’t happen in random coil) Since CTD and WT similar with little changes in tertary structure around the trp and tyr residues, CTD contributing most to tertiary structure Even though NTD is unstructured and has no signal by itself, it does contribute to tertiary structure because CTD and WT still have differences Step 3: Find delta e 192 for WT: to determine intensity have MRE in far UV WT MRE at 192nm =15000 deg cm2/dmol Delta e192=MRE/3300 , gives 4.5 This means that even though we have alpha helical elements it’s not the most alpha helical protien

Question 39

What are we mainly using near UV to find

Answer 39

Changes in tertiary structure based on mutation or ligand binding

Question 40

What are the practical considerations of CD

Answer 40

1. The CD spec is purged with dry N2 (g) to sweep out water and ozone which affect protien structure 2. Bandwidth must be 1 because cd is very sensitive method and looking at small changes in the differential absorbance of light , so want to lower noise as much as possible 3. Cd cuvette have strain free windows (completely optically clear, give no interefece) 4. Want abs of sample ideally <1 because light could get reabsorbed if sample too turbid 5. Need short 1.0mm path length and low concentration because amides have small extinction coefficients (e195= 7mM-1cM-1) in far uv 6. Same for trp, e 280 5.5 mM-1cM-1 so need 1.00cm (larger) path length and higher concentration in near UV 5 and 6 WHAT?