Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity

Lukas, Max; Schwidetzky, Ralph; Kunert, Anna T.; Backus, Ellen H. G.; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine; Bonn, Mischa; Meister, Konrad

doi:10.1021/acs.jpclett.0c03163

Cited by 23 publications

(41 citation statements)

References 41 publications

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“…The intensity of the O–H stretch band, and therefore the interfacial water alignment, is significantly higher at low temperatures. 42 , 44 …”

Section: Resultsmentioning

confidence: 99%

“…The process of bacterial ice nucleation at warm temperatures requires an appropriate pH value and intact INP structures. 44 Moreover, the activity of both classes of bacterial INs is strongly influenced by specific interactions with ions. These interactions are highly relevant to correctly predict the ice nucleation efficiency of bacterial INs under natural conditions (e.g., in the atmosphere).…”

Section: Discussionmentioning

confidence: 99%

“…Recent progress in ice-affinity purification methods now allows for isolating ice-binding proteins directly from natural sources and with high purity. 44 , 47 − 49 In the studies presented here, we utilized inactivated extracts from P. syringae , commercially available under the product name Snomax (Snomax Int.).…”

Section: Methodsmentioning

confidence: 99%

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Toward Understanding Bacterial Ice Nucleation

et al. 2022

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Bacterial ice nucleators (INs) are among the most effective ice nucleators known and are relevant for freezing processes in agriculture, the atmosphere, and the biosphere. Their ability to facilitate ice formation is due to specialized ice-nucleating proteins (INPs) anchored to the outer bacterial cell membrane, enabling the crystallization of water at temperatures up to −2 °C. In this Perspective, we highlight the importance of functional aggregation of INPs for the exceptionally high ice nucleation activity of bacterial ice nucleators. We emphasize that the bacterial cell membrane, as well as environmental conditions, is crucial for a precise functional INP aggregation. Interdisciplinary approaches combining high-throughput droplet freezing assays with advanced physicochemical tools and protein biochemistry are needed to link changes in protein structure or protein–water interactions with changes on the functional level.

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“…The intensity of the O–H stretch band, and therefore the interfacial water alignment, is significantly higher at low temperatures. 42 , 44 …”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Toward Understanding Bacterial Ice Nucleation

et al. 2022

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“… 2 , 7 , 17 Recent studies further showed that environmental factors (pH, salts, antifreeze proteins) can have very different effects on class A and class C INs. 11 , 18 − 20 Here, we use the high-throughput twin-plate ice nucleation assay (TINA) to investigate the ice nucleation activity of the lipids 1,2-dimyristoyl-3-trimethylammonium −propane (DPTAP), 1,2-dipalmitoyl- sn -glycero-3-phosphoglycerol (sodium counterion) (DPPG), 1,2-dipalmitoyl- sn -glycero-3-phosphorylethanolamine (chloride counterion) (DPPE), phosphatidylinositol (PI), lipopolysaccharides, and the effects of deuterated water, heat, and delipidation on the ice nucleation activity of P. syringae . 21 …”

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confidence: 99%

Membranes Are Decisive for Maximum Freezing Efficiency of Bacterial Ice Nucleators

Schwidetzky

Sudera

Backes

et al. 2021

J. Phys. Chem. Lett.

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Ice-nucleating proteins (INPs) from Pseudomonas syringae are among the most active ice nucleators known, enabling ice formation at temperatures close to the melting point of water. The working mechanisms of INPs remain elusive, but their ice nucleation activity has been proposed to depend on the ability to form large INP aggregates. Here, we provide experimental evidence that INPs alone are not sufficient to achieve maximum freezing efficiency and that intact membranes are critical. Ice nucleation measurements of phospholipids and lipopolysaccharides show that these membrane components are not part of the active nucleation site but rather enable INP assembly. Substantially improved ice nucleation by INP assemblies is observed for deuterated water, indicating stabilization of assemblies by the stronger hydrogen bonds of D 2 O. Together, these results show that the degree of order/disorder and the assembly size are critically important in determining the extent to which bacterial INPs can facilitate ice nucleation.

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“…It has been reported that the βhelix 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. We demonstrated for the first time that heat treatment causes most of the β-helix secondary structure in untreated Snomax ® to convert to a β-sheet or strand like structure using FTIR, and our microfluidic setup was able to show an associated decrease in the IN temperature.…”

Section: Discussionmentioning

confidence: 99%

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

2021

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Measurement of ice nucleation (IN) temperature of liquid solutions at sub-ambient temperatures has applications in atmospheric, water quality, food storage, protein crystallography and pharmaceutical sciences. Here we present details on the construction of a temperature-controlled microfluidic platform with multiple individually addressable temperature zones and on-chip temperature sensors for high-throughput IN studies in droplets. We developed, for the first time, automated droplet freezing detection methods in a microfluidic device, using a deep neural network (DNN) and a polarized optical method based on intensity thresholding to classify droplets without manual counting. This platform has potential applications in continuous monitoring of liquid samples consisting of aerosols to quantify their IN behavior, or in checking for contaminants in pure water. A case study of the two detection methods was performed using Snomax® (Snomax International, Englewood, CO, USA), an ideal ice nucleating particle (INP). Effects of aging and heat treatment of Snomax® were studied with Fourier transform infrared (FTIR) spectroscopy and a microfluidic platform to correlate secondary structure change of the IN protein in Snomax® to IN temperature. It was found that aging at room temperature had a mild impact on the ice nucleation ability but heat treatment at 95 °C had a more pronounced effect by reducing the ice nucleation onset temperature by more than 7 °C and flattening the overall frozen fraction curve. Results also demonstrated that our setup can generate droplets at a rate of about 1500/min and requires minimal human intervention for DNN classification.

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Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity

Cited by 23 publications

References 41 publications

Toward Understanding Bacterial Ice Nucleation

Toward Understanding Bacterial Ice Nucleation

Membranes Are Decisive for Maximum Freezing Efficiency of Bacterial Ice Nucleators

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

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