Amide proton exchange and in vivo folding

What is the amide proton exhcnage?

• NH (protein) + D₂O ⇌ ND (protein) + HDO

Proteins are not static as they change conformations. We can measure these structural fluctuations by observing the exchange of hydrogen atoms on the peptide backbone with deuterium (D₂O) from the solvent

What are monitoring methods of amide protin exchnage?

• NMR (detecting the disappearance of ¹H signals)

• Mass Spec (detecting the increase in mass as H is replaced by D)

Experimental setup for amide proton exchange

1. Isotopic labelling: bacteria (E. coli) are grown on media containing ¹⁵N ammonium salts to incorporate it into every peptide bond during protein synthesis 

2. The exchange: the labelled protein is extracted and purified, and then dissolved in a buffer made with D2O

3. Recordings of HSQC (Heteronuclear single quantum correlation) NMR spectra continue over minutes, hours, or days

   • In HSQC spectrum, ¹⁵N-¹H pair produces a specific cross-peak

   • Deuterium is NMR silent, so when an amide H is replaced by D in this method to become ¹⁵N-D, the signal for that specific residue disappears

4. Intensity analysis: the decrease in intensity (volume) of each cross-peak over time is analysed and interpreted:

   • Fast disappearance: residues are on the surface or in flexible loops, and are not protected by hydrogen bonds

   • Slow disappearance: residues are buried in the hydrophobic core or locked in an alpha-helix/ beta-sheet, so they only exchange when the protein changes conformation/unfolds locally

5. Fit the curve to a single exponential decay to calculate k_ex using 𝑦 = 𝐴𝑒^−𝑘_ext

What happens to the structure of the protein during the 3 main ways for the amide proton to be exposed?

1. Global unfolding (top pic): entire protein cooperative tertiary network completely collapses, opening up the deeply buried core hydrophobic regions to the solvent, exposing almost all backbone amide positions simultaneously.

2. Partial unfolding: specific domains or standalone sub-structures fold/unfold independently while the remaining body of the protein stays intact

3. Local fluctuation (bottom pic): protein maintains its overall native fold, but experiences rapid, low-amplitude thermal vibrations/breathing actions. This breaks hydrogen bonds locally, giving solvent molecules momentary access to local amides without altering the macroscopic structure

What are chaperones? Give examples

Specialised proteins that prevent aggregation and assist in the folding process of other proteins without being part of the final structure.

• HSP70 system

• Chaperoning (e.g., GroEL/GroES)

HSP70 system

• Binds to exposed hydrophobic segments of unfolded polypeptides

• Prevents aggregation

• Uses ATP hydrolysis cycle to release substrate and allow folding attempts

Chaperoning (e.g., GroEL/GroES)

• Large barrel-shaped complex

  • GroEL: a double-ring structure that captures the unfolded protein 

  • GroES: the lid that closes the cage

• Encapsulates unfolded protein in a protected chamber, allowing it to fold in isolation, this process is ATP-dependent

• Especially important for β-sheet proteins