Chemical Interactions, Purification, and Crystallisation

Question 1

4 methods of macromolecular crystallisation: and brief descriptions of them.

Answer 1

1. Batch Method (Brings the protein directly into the nucleation zone via mixing with precipitant (e.g. NACl). Usually performed using a small amount of oil to prevent evaporation)
2. Dialysis (Utilizes diffusion and equilibration of precipitant molecules through a semi-permeable membrane as a means of slowly approaching the concentration at which the macromolecule crystallizes.)
3. Liquid-Liquid diffusion (Sample is placed in a capillary and, without mixing, a precipitant solution is
   placed in contact with it. Interface diffusion occurs and promotes precipitation )
4. Vapour diffusion (Most common. Droplets containing purified protein, buffer, and precipitant are allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. “Hanging Drop” or “Sitting Drop”)

Question 2

Why are macromolecular crystallisation methods needed?

Answer 2

Macromolecules are of greater complexity and are more flexible than small molecules and so are more difficult to crystallise. They don’t crystallise by chance like small molecules.

Question 3

Chromatography Purification Methods

Answer 3

1. Ion Exchange: Separated by charge
2. Size Exclusion: Separated by size and shape
3. Affinity Chromatography: Separated by chemical binding
4. Hydrophobic Interaction : Separated by surface hydrophobicity

Question 4

2 common tags used in affinity chromatography

Answer 4

GST tags and His tags

Glutathione S-transferase) and (Histidine

Question 5

Isoelectric point (pI)

Answer 5

The pH at which a molecule carries no net electrical charge. It is least soluble at its isoelectric point

Question 6

Size Exclusion Chromatography

Answer 6

Smaller molecules spend more time in pores, larger molecules move through faster and have a small retention time

Question 7

Methods to check purity levels

Answer 7

SDS-PAGE, Mass spectrometry, Bradford assay, etc.

Question 8

General Overview

Answer 8

Plan experimental approach
Recombinant DNA methods to generate samples
Purification
Assess levels of purity
Crystallography

Question 9

Simple Crystal Description

Answer 9

An ordered array of atoms or molecules

Question 10

Crystallography

Answer 10

To generate crystals, must achieve “supersaturation”, the formation of crystal or precipitate nuclei which grows until the system reaches equilibrium (Like rolling a snowball down a hill)

Supersaturation:

• Metastable Zone
• Nucleation Zone
• Precipitation Zone