Jan 23 Flashcards
(20 cards)
What does CD measure?
It uses electronic transitions in the UV range to detect the secondary or tertiary structure of a protein, measures absorbance of the L and R handed components of light
When use near and far UV for CD?
Far UV is 178-260nm, near is 260-320 nm. Far is for amides of the protein backbone (secondary structure), near is for the tertiary structure, it uses Tyr, Phe, and some Trp to relate to environment in tertiary structure
On energy diagram, what are the orbitals that we have to know from lowest to highest energy?
From lowest to highest energy, have sigma, pi, nonbonding, then pi antiboding, then sigma antibonding
What does n to pi antibonding transition in CD measure?
It is nonbonding to pi antibonding, this less of an energy change than from pi to pi antibonding. Looks at lone pairs not bonding to anything
What do pi to pi antibonding transitions measure?
They look at lone pairs beside an aromatic
What is polarized light?
It is light that has the electric field vector in the same plane, not random
Why do chromophores absorb polarized light?
If it has a dipole moment that aligns with the electric field vector, then more light is absorbed
What is plane vs circular polarized light?
Plane will have amplidue changing on constant axis, and circular will have constant amplitude that rotates between axes
Is left circular polarized light L-CPL and R-CPL which directions ccw and cw?
L is CCW, R is CW, viewed down direction of propagation
What is the layout for CD spectrophotometry?
Have light source, usually Xenon lamp. Then monochromator, circular polarizer, sample, detector, the electonics. The circular polarizer will alternate rapidly between L and R CPR at a set frequency
What is abs vs CD spectra diffs?
Abs is molar extinction coeff on y axis, wavelength on x. Get all pos peaks
CD is change in molar extinction coeff on y axis, and wavelength on x. Can have neg and pos peaks
What is molar ellipticity vs mean residue ellipticity? When is one more used?
Molar ellipticity is the degreesx cm^2/decimol at a particular wavelength.
MRE is the average residue weight used
MRE for secondary structure
Molar ellipticity for tertiary structure
What are the rough features of a-helix, B-sheet, random coil on CD spectra?
a helix is peak at 190 nm, then double dip from 200-220 nm. B-sheet is broader peak at 200 nm, then single dip at 215 nm. Random is neg and then slight pos at 200 nm.
Why do you get double dip for a-helices?
It is because of chromophore coupling. The pi to pi antibonding transition is split because of this, so the pos peak and first neg hump is from this. The second hump from double dip is the non bonding to pi antibonding transition.
What is the threshold for a majorly a-helical protein, what change in extinction coeff value needed at what wavelength?
Need at least 10 M-1, cm-1 at 192 nm peak for it to be considered mostly a-helical
The CD of a protein at all wavelengths is the ___1____ of the CD from each of its _______2________.
1=sum
2= component secondary structures
Can have antiparallel and parallel B sheet, turns, helices, etc.
What is basis spectra and the problems with it?
It is when you know for sure that a protein is only a-helical/B-sheet etc. You use diff wavvelgnth ranges to produce a CD spectra. These values will give a rough value (0 to 1) giving how much of the protein is a-helical.
Problems are that you assume that only amides contribute, not the case, aromatics contribute a bit in far uv as well.
Why do we detect aromatic side chains with near uv light CD? How use this to get tertiary structure info?
These aromatic groups are not chiral, but act chiral if there is disymmetric surroundings. The aromatic group may have an axis of symmetry, but has no planes or points of symmetry.
From denaturation, ligand binding, subunit interaction etc. you get the tertiary structure information.
What are CD practical considerations (6)?
- Need to purge with N2 to remove H2O and ozone
- Use narrow bandpass (1 nm) to reduce noise
- Use optically clear cuvettes
- Need to have Abs smaller than 1
- Amides in far uv use short pathlength (1 mm) and small concentration
- Near UV we use larger pathlength (1 cm) and higher concentration
