JW - Protein Architecture IV

Question 1

What does chemical shift dispersion indicate?

Answer 1

Presence or absence of 3D structure

Question 2

How are correlations between two proteins used? (2)

Answer 2

• If two protons are close in space (<5A) they show a correlation peak
• If two protons are not close then no correlation is observed

Question 3

Explain the process of converting proximity information of proton pairs into 3D structure using NMR

Answer 3

Proton proximity information is converted into 3D structure by triangulation;

• if H1 is close to H2 and H2 is close to H3, then H1 is inferred to be close to H3,

Leads to the construction of a family of structurally similar structures

Question 4

What are strengths (2) and limitations (1) of Negative stain EM?

Answer 4

(sample coated in metal ions enhance contrast)

Strength:

• small amounts of sample (microg),
• image wide range of samples (proteins, lipids, large assemblies, cellular substructures)

Limitations: resolution ~20Å, artefacts through staining

Question 5

What are strengths (4) and limitations (1) of Cryo EM?

Answer 5

(sample frozen in vitreous ice)

Strengths:

• small amounts of sample (mg)
• image wide range of samples
• no staining necessary (less artefacts)
• 3D reconstruction

Limitations:

• Resolution depends on averaging techniques

Question 6

What are strengths (2) and limitations (1) of EM diffraction?

Answer 6

Strengths:

• Requires (only) 2D crystals (e.g. good for membrane proteins)
• Resolution ~ 3-4Å possible

Limitations

• EM always struggling with radiation damage.

Question 7

What are strengths (3) and limitations (4) of Scattering?

Answer 7

Strengths:

• applicable to large range of macromolecules - Proteins, DNA/RNA, small to large complexes, and assemblies
• measurement in solution
• ~mg of pure protein required

Limitations:

• Resolution ~20Å,
• Only shape information,
• best suited in combination with other techniques
• Neutron scattering requires more material and best suited with some level of deuteration (i.e. recombinant protein production)

Question 8

What are strengths (3) and limitations (3) of x-ray crystallography?

Answer 8

Strengths:

• highest resolution (less then 1Å)
• enormous information content: do chemistry by structure
• applicable to large range of macromolecules- Proteins, DNA/RNA, small to large complexes, and assemblies

Limitations:

1) Molecule needs to form crystals

• may prove impossible
• Crystal environment may influence the result of dynamic parts of a protein or weakly interacting systems

2) Static structure

3) Several mg of pure protein required

Question 9

What are strengths (9) and limitations (2) of NMR?

Answer 9

Strengths:

• Versatile tool to investigate any macromolecule- Each atom a potential “spy” to investigate a protein
• Experiments in solution or in solids (membranes, aggregates)
• Structure determination
• folding,
• Protein ligand interactions with local resolution
• dynamics (site specific with out perturbation of protein)
• highest resolution (less then 1Å)
• enormous information content: do chemistry by structure
• applicable to large range of macromolecules

Limitations:

• Several mg of pure protein required (isotope labelling , recombinant protein production
• Solutions structures limited to small (<50Kda) proteins