Structure Determination

Question 1

Circular dichroism

Answer 1

a spectroscopy technique that measures the absorption difference between left and right circularly polarized light

Question 2

Enantiomers

Answer 2

optical isomers/ mirror images
rotate plane polarised light

Question 3

Circular dichroism- use?

Answer 3

CD spectra provides information on
secondary structure
AA have polarised waves => orientation of secondary structure depends on protein spectra

Question 4

X-ray Crystallography

Answer 4

Gives atomic resolution, needs crystals, structure is effected by crystal lattice, a single
conformation.

wavelength: 10^-10m

crystal- diffraction pattern- electron density map- protein model

Question 5

Diffraction and interferance

Answer 5

process whereby electromagnetic
radiation is scattered by matter (here, slit of
width of one wavelength)

Question 6

Bragg’s law

Answer 6

Bragg’s law provides a
measure of distance between
planes
nλ = 2 d sin(θ) (Bragg’s law)

Question 7

resolution

Answer 7

Higher resolution= higher confidence in model

higher res meand smaller A value

Question 8

Nuclear Magnetic Resonance

Answer 8

NMR is based on application of
magnetic field and measuring energy required for making transition between two spin
states.

Question 9

nuclei consisting of even number of particles (protons and neutrons) have a

Answer 9

non-zero spin and as a result develop a magnetic field

Question 10

Advantages of nmr over X-ray

Answer 10

Structure in solution
Provides information on multiple conformations
Requires large amount of very pure protein (miligrams)

Question 11

limitation of NMR

Answer 11

Can be applied only to relatively small proteins

Question 12

Cryo Electron Microscopy (CryoEM):

Answer 12

used to determine the 3D structures of biomacromolecules- sensitive

High resolution up to atomic, can be used on crystals and ordered assemblies of isolated
particles, can trap transient states (multiple conformations).

Question 13

Franks image analysis for 3D structures

Answer 13

1. electron beam hits randomly oriented proteins leaving a trace image
2. computer discriminates between traces and fuzzy background and stacks similar images
3. stacks of 1000s, 2D high resolution image genearted
4. computer calculates how the different 2D images relate to each other and a high- resolution 3D structure formed