Topic 5

Question 1

• As protein size …, ¹H-¹⁵N/¹H-¹³C … experiments become … and difficult to interpret.
• Can add additional …-… to further delineate residues based on … chemical shift, giving … resonant experiments.
• E.g. a 3D HNCA experiment correlates H,N and Cα, where any α-carbons, will be directly above a corresponding HN shift

Answer 1

• As protein size increases, ¹H-¹⁵N/¹H-¹⁵N hybrid experiments become crowded and difficult to interpret.
• Can add additional ¹³C-filter to further delineate residues based on ¹³C chemical shift, giving triple resonant experiments.
• E.g. a 3D HNCA experiment correlates H,N and Cα, where any α-carbons, will be directly above a corresponding HN shift

Question 2

Describe the CBCANH 3D correlation method

Answer 2

• C_α are positive
• Does not tell us which residue is i and which is i-1

Question 3

Describe the CBCA(CO)NH 3D correlation method

Answer 3

• Always paired with CBCANH, to determine difference between residues i/i+1
• Strips of these resolved peaks can be used to sequentially assign backbone (not identity of AA though)
• Allows magnetisation only through C=O carbon

Question 4

Describe the HNCA/HN(CO)CA pair that could be run to reinforce speculations made so far.

Answer 4

• HNCA: In each NH strip, 2 C_α peaks visible from residue i and i+1
• HN(CO)CA: In each NH strip, 1 C_α visible from preceding residue (i-1)

Question 5

Describe the HNCO/HN(CA)CO pair that could be run to reinforce speculations made so far.

Answer 5

• HNCO: In each NH strip, 1 CO visible from residue i-1
• HN(CA)CO: In each NH strip, 2CO peaks visible from residue i and i-1

Question 6

• What 2D experiments should be done to assign side chains of our protein first?

Answer 6

• HCCH-TOCSY: At each ¹³C chemical shift, see ¹H atoms attached to given C and all other Cs in side chain
• HCCH-COSY: At each ¹³C chemical shift, see ¹H atoms attached to given C and neighbouring Cs only.
• Nitrogen methods not used as not present in most sidechains

Question 7

What additional experiments can be done to assign side chains of our protein?

Answer 7

• Use CC(CO)NH and H(CCO)NH in combination with our HCCH-TOCSY/COSY
• CC(CO)NH: in each NH strip, see all C nuclei in sidechain of residue i-1
• H(CCO)NH: in each NH strip see, all H nuclei in sidechain of residue i-1

Question 8

How does sensitivity vary throughout all the experiments described?

Answer 8

• HNCA/HNCO have high sensitivity, despite less chemical information being given as a result of less filtering
• HNCA gives poor resolution in 3D due to signal loss through added filter via relaxation effects.
• Both have high signal:noise ratio, due to lots of acquisitions occurring throughout 3D data generation
• 2D HSQC therefore takes a lot less time than these long 3D experiments.

Question 9

For proteins >25kDa, the methods discussed so far do not suffice. What is this due to and how can we get around it?

Answer 9

• Broadening in peaks as we move to higher mass systems is the result of a shorter T₂ associated with spin-spin relaxation, making resolution unmanageably poor to take in to 3D.
• All Hs interacting due to spin-spin relaxation as well as the facilitation of spin relaxation of neighbouring C/N atoms is a problem that increases with molecular weight.
• Circumvent this by deuterating our sample, reducing the proton density, slowing relaxation due to dipole-dipole interactions and CSA of quadrupolar deuterium

Question 10

Deuteration can be combined with TROSY to determine structure of large proteins, what is TROSY and how does it do this?

Answer 10

• Transverse relaxation optimized spectroscopy selects the sharpest component where, DD and CSA relaxation mechanisms cancel
• Less overlapping in this peak, allowing increased spectral resolution
• Increases sensitivity
• Extends mass range

Question 11

As well as uniform isotopic labelling, site-selective labelling can be employed, what is this? give a few examples

Answer 11

• expensive/time consuming process
• gives spectral simplification
• good probe for structrual/dynamics studies
• sequence-specific resonance assignments for peaks in crowded regions
• amino acid selective labelling
• methyl-specific protonation

Question 12

If a protein is unassignable, what is an alternative method that could be used to understand ligand binding dynamics

Answer 12

• structural assignment not goal, so can use STD NMR to investigate binding constant of ligand binding
• Titration series of increasing enzyme concentration done where change in intensity of ligand signals detected via NOE
• Amplification factor calcuated at each [ligand] - describes how much peak impacted by ligand presence.

Question 13

What response is generated from plotting STD AF values vs [ligand] in STD NMR

Answer 13

Michaelis-Menten hyperbolic dose-response