Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Authors

Bhuvanendran, H., Brunner, C. M., Kempf, H., Moro, J. L., Roubieu, E., Turbant, F., Mateus, A., Lin, H., Das, L., Malyshev, D., Johns, B., Parracino, A., Pastore, A., Peters, J., Cortajarena, A. L., Zanetti Polzi, L., Maccaferri, N.

Abstract

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 27 Jun 2026.

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