Combining two-dimensional infrared spectroscopy (2D-IR) and vibrational sum-frequency generation (VSFG) with spectral calculations based on molecular-dynamics (MD) simulations opens up new avenues to study molecular processes. The highly structure- and bulk-sensitive 2D-IR spectra can be related to the surface-sensitive VSFG spectra by excitonic calculations in order to resolve structural changes as a function of environment (solution versus interface), to thus reveal the importance of interfaces in molecular mechanisms. We have employed this combination of techniques to study how ice-nucleating proteins (INPs) perform their function. INPs are exposed on the membranes of ice-nucleation active (INA) bacteria, which can promote the growth of ice more effectively than any other known material. These bacteria obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, drive ice nucleation within clouds, which may affect global precipitation patterns. Despite the evident environmental importance of this process, the molecular mechanism behind INP-induced freezing have remained largely elusive. The development of a novel INP construct allows us to directly investigate the structural basis of the interactions between water and the ice-nucleating protein ‘InaZ’ from the INA bacterium Pseudomonas syringae, for the first time. With 2D-IR spectroscopy in the amide-I region, we determine that the ice-active repeats of InaZ adopt a β-helical fold in solution, and with VSFG spectroscopy we show that this conformation is contained at air–water surfaces (see figure). VSFG spectra of the OD-stretching modes reveal highly ordered interfacial water species, with an increased ordering when the interface is cooled to temperatures close to the melting point of water, the ‘operating temperature’ of the bacterium. The VSFG data, combined with MD simulations and spectral calculations, indicates that INP reorients at lower temperatures. This reorientation can enhance water interactions, which are likely to affect the effectiveness of ice nucleation.

Revealing the molecular structure and mechanism behind bacterial ice-nucleation with vibrational sum-frequency generation and 2D-IR spectroscopy

ACS Spring Meeting 2021

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