Topic 1: What is Structural biology

Question 1

What is meant by structural biology? (dntk)

Answer 1

Concerns how we understand biomolecular function through characterisation of structure, interactions and dynamics.

Question 2

What is the structure-function hypothesis?(dntk)

Answer 2

• Each molecule encoded by a genome has function.
• Function only possible after coiling into specific 3D shape
• This must be done so lightly, and efficiently

Question 3

Why do we care about structural biology?(dntk)

Answer 3

• Inform us on how different molecular architectures (tertiary structure) perform in chemical reactions

Question 4

What experimental techniques are married to structural biology? (dntk)

Answer 4

• Mass spec
• X-ray diffraction
• NMR
• Computational methods

Question 5

What problems can be explored in structural biology? (dntk)

Answer 5

• Biomolecular self-assembly
• Biomolecular dynamics
• Structure-activity relationships
• Biomolecular interactions
• Biomolecular structure
• Ligand binding/design
• Physical chemistry of biomolecules (e.g. thermodynamic properties)

Question 6

Why is knowing about biomolecular dynamics important? (dntk)

Answer 6

• Dynamics impact function and binding, so must know all the states of moving biomolecules that may be hidden using NMR

Question 7

Why is understanding the structure-activity relationship of a protein important?

Answer 7

• Adoption of correct active structure important for biomolecular function or genome will get rid of any unneeded protein.
• Protein mis-folding is believed to be the result of amyloid disease associated with Alzheimer’s.

Question 8

• Structure of a biomolecule allows it to recognize … partners
• … events are on/off processes that can trigger a … , all starting with a few key interactions that are dependent on …

Answer 8

• Structure of a biomolecule allows it to recognize binding partners
• Binding events are on/off processes that can trigger a signal, all starting with a few key interactions that are dependent on structure

Question 9

What is a method we can use to help characterise biomolecular interactions?

Answer 9

• Isotopic labelling can be used to enrich the system with ¹³C/¹⁵N.
• As these are low abundance, they will stand out in analysis.

Question 10

Why is understanding ligand binding and design important? (dntk)

Answer 10

Knowledge of molecular determinants of ligand binding allows refinement of existing or design of new ligands/drugs that can impact function

Question 11

• What is the size limitation problem in solution NMR? How is solid state affected by this?(IMP)

Answer 11

• Rotational correlation time (τ_c) scales inversely with MW
• As MW ↑, T₂ signal-to-noise ↓ and peaks broaden
• This is due to T₂ being so short that signals relax quicker than data can be acquired.
• Solid state NMR does not suffer from this, however peaks are generally broader anyway

Question 12

• What is the variable intensity problem? (IMP)

Answer 12

• Intermediate exchange regime leads to variable peak intensities
• meaning some peaks will be weak, others intense
• 𝛿_A = resonant frequency associated with state A
• Larmor frequency difference (Δω) >> rate of exchange between A/B (k_ex) à see both states
• Δω << (k_ex) forms single weighted average state
• Δω ≈ (k_ex) results in broadening –> variable exchange

Question 13

What is the problem associated with proteins unfolding? What conditions can cause this to occur?

Answer 13

• Aggregation on proteins can lead to broad peaks
• Poor chemical shift dispersion of ¹H peaks leading to severe overlap, and will appear all in the same environment
• High pressure can cause proteins to unfold and chemical shift envelope to become more compact

Question 14

What is insensitivity in NMR? How can it be overcome?

Answer 14

• 1 in 1E+06 signals are being measured of all excited nuclei, meaning experiments can take many days as this is very few signals (insensitive)
• This can be overcome by running at higher temperatures, but biomolecule may not be able to retain its stable fold

Question 15

Why is the knowing stability of a protein fold important?

Answer 15

Conditions in which this takes place are important to know as will massively change structure of sample

Question 16

What is the problem with isotopic labelling?

Answer 16

• Enriching our organic sample with low abundance ¹³C/¹⁵N isotopes is generally done by growing bacteria and enriching them.
• These feedstocks can be very costly.

Question 17

Advantages of NMR

• NMR provides atomic … for molecules in solution with MW ≤ 1E+05 Da and can also mirror … conditions. Doing this in solution is the best way to validate properties in a biological … ….
• NMR can be used to access … complexes in …-binding studies where … are monitored
• Accessible … nuclei cover all biologically-relevant suspects: … ¹H, ¹⁵N, …, ³¹P

Answer 17

Advantages of NMR

• NMR provides atomic resolution for molecules in solution with MW ≤ 1E+05 Da and can also mirror physiological conditions. Doing this in solution is the best way to validate properties in a biological cellular environment.
• NMR can be used to access larger complexes in ligand-binding studies where ligands are monitored
• Accessible isotopic nuclei cover all biologically-relevant suspects: ¹³C, ¹H, ¹⁵N, ¹⁷O, ³¹P