Water freezing and ice formation are fundamental processes for the environment on the planet that are regulated to a great extent by ice nucleators of biological origin. Microorganisms catalyze heterogeneous water freezing by using ice-nucleating proteins, which consist of large central units with repeating threonine motifs possibly acting as templates for water binding. Elucidating the water structure around such threonine-rich protein sites is of particular interest for revealing biological ice-nucleation mechanisms. A very promising approach for this purpose is sum frequency generation (SFG) spectroscopy that is based on frequency mixing between high-power visible and IR laser beams. SFG reveals the rich molecular structural information contained in vibrational spectra and exclusively detects species at an interface due to its selection rules.<br/>Here, we use a peptide with alternating threonine and leucine sequence (LTα14) as a simple model system to study the water structure at threonine-rich sites of ice-nucleating proteins with broadband vibrational SFG spectroscopy. We will discuss SFG data of the LTα14 peptide at the water-air interface obtained in the amide and water spectral regions. In particular, isotopic dilution experiments with heavy water indicate the presence of an additional OH mode that might be related to water interacting with threonine OH groups. Furthermore, we examine how lowering the temperature affects the water SFG spectra as well as the influence of environmental conditions such as varying pH on the water and peptide spectra. In conclusion, the data provide insights into the interaction of threonine residue-rich peptides with water and can be used to extend our understanding of the function of ice-nucleating proteins.

Probing protein and water structure at threonine-rich sites with vibrational sum-frequency generation (SFG) spectroscopy

ACS Spring Meeting 2021

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