Protein Structure and Function

Question 1

What do atoms in proteins undergo that can be detected by infra-red or Raman spectroscopes?

Answer 1

Small-scale vibrational and rotational motions.

Question 2

When do large multi-domain proteins undergo large movements?

Answer 2

Upon ligand binding

Question 3

What can proteins do spontaneously but rarely?

Answer 3

Undergo transient but complete unfolding.

Question 4

What evidence from using quenching reagents to observe protein dynamics are there?

Answer 4

That fluorescence of aromatic amino-acids is instantly quenched by close proximity of chain small molecules and that internal residue are also quenched. This suggests that the solvents can ‘dissolve’ into the protein or the protein undergo breathing motions to expose the buried regions.

Question 5

What is used to monitor hydrogen exchange?

Answer 5

Mass spectrometry or NMR. Monitors the extent of exchange versus time.

Question 6

What are protons in proteins?

Answer 6

Very sensitive to their environment.

Question 7

What is the rate of hydrogen exchange dependent on?

Answer 7

pH, location of each amide within a protein, the degree of burial from the solvent, frequency of partial unfolding and hydrogen bonding in the native state.

Question 8

Why is it not possible to use 1D hydrogen exchange for large proteins?

Answer 8

There are too many proton signals with similar chemical shift and the width of each peak increases resulting in a degenerate spectrum.

Question 9

What is used for large proteins?

Answer 9

2D hydrogen exchange. Minimises overlapping or clustering of signals by using two parameters to sort out the data.

Question 10

What two parameters are used in 2D hydrogen exchange?

Answer 10

Isoelectric focussing and SDS-PAGE.

Question 11

A fingerprint of all -NH groups except for what is generated in 2D hydrogen exchange?

Answer 11

Proline.

Question 12

What three pieces of evidence of protein dynamics were found using NMR?

Answer 12

Ring flipping, dynamic regions and a richness of dynamics from ps to seconds.

Question 13

Which residues cannot rotate their rings?

Answer 13

Tryptophan and histidine as they are too large and require 360 degree rotation.

Question 14

What two pieces of evidence of protein dynamics were found using crystallography?

Answer 14

No density and B-factors and that small proteins give different structures.

Question 15

What is the temperature factor B?

Answer 15

The B-factor which is the extent of smearing due to the local flexibility of the polypeptide. Low B-factors indicate cold, static areas and high B-factors indicate hot, dynamic areas.

Question 16

How can small proteins give different structures?

Answer 16

Crystallising at different temperatures and their relative positions of the backbone carbon on different crystal lattices.

Question 17

How are molecular dynamics analysed?

Answer 17

Atoms of known structure are given a certain velocity and a random motion. The magnitude of the force acting on each of these atoms is then calculated. As the original position and velocity is known the effect of the force can be used to calculate the new positions of all the atoms. For molecular systems the force acting on each atom is defined as a sum of terms which include bonded and non-bonded interactions.

Question 18

What information can protein dynamics reveal?

Answer 18

Highly detailed information at a molecular level, a rich pattern of atomic movements and rates of inter-conversion and relative stabilities of each conformation.

Question 19

What does roughness in the native well of an energy landscape of a protein lead to?

Answer 19

Discrete but randomly inter converting states.

Question 20

Why are protein dynamics important?

Answer 20

For multi-enzyme complexes, active site availability, ligand binding and catalysis.

Question 21

What are the two types of empirical observations that can be used to observe protein dynamics?

Answer 21

Proteins undergoing small and large scale conformational changes and oxygen release from oxyhaemoglobin.

Question 22

What are some methods of detecting macro molecular interactions?

Answer 22

Direct visualisation, genetic methods, biochemical binding assays and biophysical assays.

Question 23

Why is quantitative characterisation of binding and intermolecular interactions required?

Answer 23

To find specific vs. non-specific association, stable vs. transient complexes, strong vs. weak interactions, the type of interactions involved, the reaction between ligand/protein structure and strength and understanding of regulation of affinity by post-translational modification and solution conditions.

Question 24

What is a method of direct visualisation?

Answer 24

Using fluorescence microscopy.

Question 25

What are the problems with using fluorescence microscopy?

Answer 25

Optical resolution is limited to ~ 200 nm and a protein/complex is between ~ 2-20 nm. Slain light microscopes cannot discern between direct and indirect interaction and fortuitous co-localisation.

Question 26

What is a better method of directly visualising interaction?

Answer 26

FRET: fluorescence resonance energy transfer. Can measure between 2-8 nm.

Question 27

What has to be carried out after a pull-down assay?

Answer 27

Gel electrophoresis and a western blot.

Question 28

What are the limitations of the simple binding assay?

Answer 28

Stability of the complex during washing, detection and separation of bound ligand may be difficult, non-specific binding may completely mask specific signal, limited to low receptor concentrations and strong binding and strong signal from bound ligand is required.

Question 29

Why is a competition assay useful?

Answer 29

Enables comparison of many ligase using the same assay format- high throughput screening. Enables measurements of higher affinities compared to isothermal titration.

Question 30

What does isothermal calorimetry measure?

Answer 30

A label-free, quantitative binding assay that measure delta H directly from the heat absorbed or released. Gives a full thermodynamic description of the types of interactions involved directly to a computer.

Question 31

If delta H is above 0 what does this mean?

Answer 31

Heat is absorbed and the reaction is endothermic (and vice versa).

Question 32

Give an example of a first and second order reaction?

Answer 32

Radioactive decay is first order and ligand binding is second order.

Question 33

Briefly describe surface plasmon resonance?

Answer 33

A method to observe binding, measure kinetics, affinity, specificity and concentration without the use of ligands in a range of bio molecules from ions to viruses. Uses a glass-slide coated in a layer of gold and a prism to measure the angle produced as a light beam is projected towards the bound ligand and the flowing analyte. The angle varies dependent upon the amount of bound analyte.