Protein Dynamics

Question 1

Intrinsically disordered proteins

Answer 1

• Lack definable structure (no well-defined conformations in their
  structural ensemble)
• Often lack a hydrophobic core
• Have high densities of charged residues (Lys, Arg, Glu) and Pro
• Are not misfolded!

Question 2

Stokes radius

Answer 2

• The Stokes radius or hydrodynamic
  radius (Rh) - corresponding radius of a
  hard sphere that diffuses at the same
  rate as the protein
• In IDPs, measured values are averages
  over a large number of individual
  conformations that may differ
  substantially in size and shape

Question 3

Intrinsic disorder determinants

Answer 3

• Display both low hydrophobicity and high net charge, both of which
  prevent hydrophobic collapse

Two classes:
* Random coil (extended) states with high stokes radii
* More compact “pre-molten globules” that display stokes radii
between the canonical “molten globule” and purely extended
states

• Percentage of charged amino acids divided by the percentage of
  hydrophobic amino acids => higher values = random coil
• More than 25% of eukaryotic proteins are mostly disordered and more
  than 50% possess IDRs => unstructural biology

Question 4

Hydrogen-deuterium exchange mass
spectrometry (HDX-MS)

Answer 4

Monitor dynamics and conformational changes of a protein under native
conditions in solution

• Applications:
  
  • Study small proteins, large, complex assemblies, and intrinsically
    disordered proteins
  • Analyze structure
  • Assess conformational changes during catalytic function
  • Pinpoint binding surfaces
• Involves putting your protein of interest into a solution of D 2 O and monitoring
  incorporation of deuterium into the protein over time
  Hodge et al, 2019, Protein Science

MECH:
1. Transitions are monitored under equilibrium conditions during
continuous labeling HDX-MS experiments.
2. A protein is diluted into a deuterated solution for various amounts of
time, ranging from seconds to hours or days.
3. The exchange reaction is then quenched by dropping the solution pH to
2.5 and 0° C, where the labeling rate is at its minimum.
4. Labeled protein is digested into peptide fragments that are separated
and analyzed by liquid chromatography coupled to mass spectrometry
(LCMS).
5. LC separation must be performed quickly and under quench conditions
to limit back exchange of the deuterium label with water in solution.