Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis

Roderick P. Tas (Corresponding author), Marco M.R.M. Hendrix, Ilja K. Voets (Corresponding author)

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16 Citaten (Scopus)
88 Downloads (Pure)


Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

Originele taal-2Engels
Aantal pagina's8
TijdschriftProceedings of the National Academy of Sciences of the United States of America (PNAS)
Nummer van het tijdschrift2
StatusGepubliceerd - 10 jan. 2023


ACKNOWLEDGMENTS. We thank A. Aloi for initiating the construction of the cooling stage and objective collar and J. Grimm and L. Lavis at Janelia Research Campus for kindly gifting HaloLigand-paJF646 and E. Katrukha for the script to import DoM data into the msdanalyzer Matlab class. This work is supported by the Dutch Research Council to R.P.T (NWO-VENI: 202.220, understanding ice growth inhibition by AFPs through super-resolution microscopy) and the European Research Council to I.K.V (ERC-2020-CoG 101001965).

European Research CouncilERC-2020-CoG 101001965
Nederlandse Organisatie voor Wetenschappelijk Onderzoek202.220


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