The Ice Nucleating Protein InaZ is Activated by Low Temperature

Abstract

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Utilizing specialized ice-nucleating proteins (INPros), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds and may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INProinduced freezing have remained largely elusive. In the present study, we investigated the folding and the structural basis for interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae strain R10.79. Using vibrational sum-frequency generation and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces.In this configuration, hydrogen bonding between INPros and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with spectral calculations and molecular-dynamics simulations shows that the INPro reorients at lower temperatures. We suggest that the reorientation can enhance order-inducing water interactions and, thereby, the effectiveness of ice nucleation by InaZ.

Toward Understanding Bacterial Ice Nucleation

“… 43 Shortened INPs with low ice nucleation activity expressed in E. coli showed a similar effect, and the observation was attributed to an activation of INPs at lower temperature and the ability to order water, which increases close to the respective freezing temperature. 45 While providing much needed experimental insights into the INP/water interface, these studies and interpretations must be taken with a caveat, given that more recently it has been shown that water ordering at lower temperatures observed with SFG ( Figure 3 C) are identical for active INPs and heat-denatured INPs that have completely lost their ice nucleation activity.…”

A Microfluidic Device for Automated High Throughput Detection of Ice Nucleation of Snomax®

“…We also measured the secondary structure of the ice nucleating protein inaZ which is primarily responsible for templating water molecules to form ice and lowering the nucleation barrier [ 69 , 70 ]. It has been reported that the β-helix region of this protein is the primary IN site [ 71 , 72 ] and its presence can be detected using FTIR techniques [ 73 ] from the amide—I region of its IR spectra. A study into the amide—I region of purified IN proteins from Snomax ® was published recently [ 74 ], where the authors concluded that heating above 55 °C caused irreversible changes to the protein structure which resulted in reduced IN activity.…”

Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity

“…Despite their multifaceted environmental impacts, only a limited number of modelling-and experimental studies on the structures and interactions of bacterial INpro has been carried out (Garnham et al, 2011b;Vanderveer et al, 2014;Pandey et al, 2016;Hudait et al, 2018;Ling et al, 2018;Zee et al, 2019;Roeters et al, 2020;Lukas et al, 2021;Schwidetzky et al, 2021a). For example, based on a homology modelling study, it has been suggested that each of the 16 amino acids repeats forms a right-handed β-helix with a triangular base and three β-sheet faces, which aligns the threonine-x-threonine motifs (TxT motif) along one face of the β-helix and the serine-xleucine-threonine/isoleucine (SxL[T/I] motif) along the other face, with each motif forming a short β-strand.…”

“…Using interface-specific sum frequency generation (SFG) spectroscopy in combination with modelling, Pandey et al (2016) proposed that the extended domain of aligned TxT motifs enhances ice-nucleation through a hydrophilichydrophobic pattern (Pandey et al, 2016). Recently, data from a combined SFG and two-dimensional infrared spectroscopy study (Roeters et al, 2020) were shown to agree better with a newer β-helical INpro structure (Garnham et al, 2011b) over an older β-hairpin model (Kajava and Lindow, 1993). They also showed that the protein imposes order on adjacent water molecules at low temperature (Roeters et al, 2021) but this has been shown insufficient to explain the INpro activity (Lukas et al, 2021).…”