A brief introduction to HX

Hydrogen exchange

In the hydrogen exchange (HX) experiment, we measured the rate of hydrogen (H) exchange for deuterium (D) in the backbone amide groups of proteins at equilibrium. The H-D exchange reaction occurs when transient opening of the protein backbone exposes the amide group to solvent.

\[\text{NH(closed)} \underset{k_{\mathrm{cl}}}{\stackrel{k_{\mathrm{op}}}{\rightleftharpoons}} \text{NH(open)} \stackrel{k_{\mathrm{ch}}}{\longrightarrow} \text{exchanged}\]

All canonical amino acids except for prolines are involved in backbone amide H bonds in the protein structure and can therefore be measured in the experiment.

Exchange rate

Under the steady-state approximation:

\[k_{\mathrm{ex}} = \frac{k_{\mathrm{ch}} \cdot k_{\mathrm{op}}}{k_{\mathrm{ch}} + k_{\mathrm{cl}} + k_{\mathrm{op}}} \approx \frac{k_{\mathrm{ch}} \cdot k_{\mathrm{op}}}{k_{\mathrm{ch}} + k_{\mathrm{cl}}}\]

In this reaction, K_op, defined as k_op/k_cl, is the opening equilibrium constant. k_ch is the intrinsic or chemical exchange rate for the amino acid in the context of its neighbors in an unstructured polypeptide. The k_ch rates were previously experimentally determined.

EX2 regime

In folded proteins at equilibrium, the hydrogen exchange reaction occurs as the protein rapidly unfolds and refolds or experiences local fluctuations in the conformational ensemble, allowing each hydrogen to exchange individually. In this EX2 regime, k_cl » k_ch because the fold prevents immediate rapid hydrogen exchange.

K_op and k_ch are related by the H-D exchange rate, k_ex, that is observed in the experiment at the EX2 regime (where K_op « 1, favoring the closed state).

In the EX2 regime, therefore:

\[k_{\mathrm{ex}} = K_{\mathrm{op}} \cdot k_{\mathrm{ch}}\]

Free energy of opening

With knowledge of these terms, we can determine the free energy of opening, ΔG_op, for each amino acid:

\[\Delta G_{\mathrm{op}} = -RT\ln(K_{\mathrm{op}}) = RT\ln(k_{\mathrm{ch}}/k_{\mathrm{ex}})\]

The equilibrium constant K_op or the ratio k_ch/k_ex is also known as the protection factor (PF).

Summarized from 1,2,3,4

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References

1. Bai, Y., Sosnick, T., Mayne, L. & Englander, S. (1995). Protein folding intermediates: native-state hydrogen exchange. Science, 269, 192–197.
2. Chamberlain, A., Handel, T. & Marqusee, S. (1996). Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH. Nature structural biology, 3, 782–787.
3. Hollien, J. & Marqusee, S. (1999). Structural distribution of stability in a thermophilic enzyme. Proceedings of the National Academy of Sciences, 96, 13674–13678.
4. Lu, C., Wells, M., Reckers, A., McBride, S. & Glasgow, A. (2026). Site-resolved energetic information from HX-MS experiments. Nature Chemical Biology, 22, 307–317.