MolBio2 - 45

Question 1

Define K-off[AB]

Answer 1

Rate of dissociation of AB into A+B

Question 2

Define K-on[A+B]

Answer 2

Rate of association of A+B into AB

Question 3

Define protein interaction equilibrium

Answer 3

Association rate = dissociation rate; K-on = K-off

Question 4

Define the protein interaction equilibrium constant

Answer 4

K-on / K-off

Question 5

List 5 major methods of protein binding detection

Answer 5

ELISA, PAGE, Western Blot, Far Western Blot, Surface Plasmon Resonance

Question 6

What does ELISA stand for?

Answer 6

Enzyme-linked immunosobrent agent

Question 7

What does PAGE stand for?

Answer 7

Poly-acrylamide gel electrophoresis

Question 8

What is surface plasmon resonance also known as?

Answer 8

Biacore

Question 9

Draw how Biacore works

Answer 9

Question 10

Describe how surface plasmon resonance works.

Answer 10

Bait molecule added to gold film by flexible tether, and inserted. Solution of prey molecules passes by, binding to some bait molecules. Binding causes measurable alterations in the plasmon resonance field.

Question 11

What is plasmon resonance?

Answer 11

Magnetic field close to the gold strip, setup by incident light

Question 12

What are the 2 key disadvantages to surface plasmon resonance?

Answer 12

Tether can block binding site, gold can affect the bait protein

Question 13

List 4 methods of protein structure detection

Answer 13

Circular dichroism spectroscopy, x-ray crystallography, NMR, EM

Question 14

What is circular dichoism?

Answer 14

Profile of the differences in absoprtion of left and right-handed polarised light

Question 15

What does CD stand for?

Answer 15

Circular Dichroism

Question 16

What spectral region of CD reveals protein secondary structure?

Answer 16

Far-UV - 190-250nm

Question 17

What three types of protein secondary structure are there?

Answer 17

Beta-pleated sheet, alpha-helix and random coil

Question 18

What does CD spectroscopy tell us about proteins?

Answer 18

% proportion of each kind of secondary structure, but no information on arrangement

Question 19

What spectral region of CD reveals protein teriary structure?

Answer 19

Near-UV - 250-350nm

Question 20

What particular signals are shown on teriary structure CD spectroscopy?

Answer 20

Aromatic amino acids and disulfide bonds

Question 21

Why are aromatic acids and disulfide bonds particularly sensitive to CD spectroscopy?

Answer 21

Their absorbance is affected by the local environment and can be observed dynamically

Question 22

What pre-requisites are there to x-ray crystallography?

Answer 22

Proteins need to be highly pure and in crystal form

Question 23

Describe the mechnism of x-ray crystallography

Answer 23

High energy and focussed beam mof x-rays fired through protein crystal. Most pass straight through, some deflected giving diffraction pattern

Question 24

How is the strucure of a protein determined from an x-ray crystallograph?

Answer 24

Structure traced back from diffraction pattern

Question 25

What is the resolution of an x-ray crystallograph dependent on?

Answer 25

How high energy the x-ray is and how fine the detector is

Question 26

What molecules are used in NMR?

Answer 26

H1, C13, N15

Question 27

How does NMR work?

Answer 27

NMR active nuclei resonate at specific frequencies in magnetic fields. Dependent on local environment, different protons resonate at different frequencies - chemical shift

Question 28

What is chemical shift?

Answer 28

Difference in proton resonation in NMR dependent on local environment

Question 29

How are proteins generated for NMR?

Answer 29

Recombinantly in bacteria with only C13/N15 available, so they are NMR-active

Question 30

Describe NMR structure results

Answer 30

Iterative process, so several possible structures are represented as an ensemble for analysis

Question 31

How are specimens preserved in EM?

Answer 31

Negative stain or vitreous ice (cryo-EM)

Question 32

What kinds of structure are best for EM?

Answer 32

More symmetrical and ordered leads to easier averaging process

Question 33

What are the slowest protein structure analysis methods?

Answer 33

Crystallography - growing crystals; NMR - analysis

Question 34

What are the quickest protein structure analysis methods?

Answer 34

Solution biochemistry or CD

Question 35

What are the most expensive protein structure analysis methods?

Answer 35

Crystallography and NMR

Question 36

What is the cheapest protein structure analysis method?

Answer 36

Solution biochemistry

Question 37

What protein structure analysis method has the highest resolution?

Answer 37

Crystallography

Question 38

What protein structure analysis method has the lowest resolution?

Answer 38

Solution biochemistry/CD - affinities/secondary structure only

Question 39

What protein structure analysis methods have no limit on protein size?

Answer 39

Solution biochemistry, CD, crystallography

Question 40

What is the limit on NMR protein sample sizes?

Answer 40

50kDa

Question 41

What protein structure analysis methods require only small samples?

Answer 41

Solution biochem, CD, EM (to some extent)

Question 42

What protein structure analysis methods require large sample sizes?

Answer 42

Crystallography, NMR

Question 43

What are the possible problems with crystallography?

Answer 43

Can have crystallisation artefacts and dynamics analysis is limited

Question 44

What are the disadvantages of NMR?

Answer 44

Need very high concentrations of sample AND isotopic labels, size limitation

Question 45

What is a key limitation in EM analysis of proteins?

Answer 45

No dynamics