Fluorescence Microscopy Evidence for Quasi-Permanent Attachment of Antifreeze Proteins to Ice Surfaces

Abstract

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.

Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

“…These surface densities correspond to distances between GFP-TmAFP molecules ranging from 6 nm to 18 nm. These values of surface density are higher than those previously reported for AFGP and type III AFP (9,22).…”

“…2C, green line), indicating that the GFP-TmAFP molecules bound to the ice crystal remained adsorbed over the course of the medium exchange process. We estimated the density of the GFP-TmAFP molecules on the ice surfaces from measurements of fluorescence intensity in the solution and at the ice surfaces, as described previously (22). We found surface densities ranging from 3,000 to 25,000 molecules per square micrometer, depending on the concentration in solution, plane of adsorption examined, and time allowed for accumulation.…”

“…. The last set of data points represents the fluorescence intensity of GFP-TmAFPs adsorbed on the ice surface (Ice) and is calculated from the two first datasets, Solution and Edge, respectively, using the equation Ice = Edge − Const × Solution, where Const is a fitting parameter, which reflects the contribution of GFP-TmAFPs in solution into the fluorescence at the edge of the ice crystal (22). Fig.…”

“…Thus, the authors concluded that the binding of these proteins to the ice surface was weak. In contrast to the above results, Pertaya et al (22), who used fish AFP type III (23) tagged with green fluorescent protein (GFP) and the technique of fluorescence recovery after photobleaching (FRAP), found the binding of AFPs to ice surfaces to be irreversible. Nevertheless, these experiments did not directly address the question of the influence of AFPs in solution on the FH activity.…”

Superheating of ice crystals in antifreeze protein solutions

“…The experimental cell used in the fluorescence microscopy experiments was the same as that used in the nanoliter osmometer experiments, except that the samples were sandwiched between two cover glasses rather than placed in immersion oil. The cover glasses were sealed together with polydimethylsiloxane (Sylgard 184, Dow Corning Corp.) which was prepared with a ratio of 1∶10 [vol/vol] between the curing agent and the base (41). The sandwiched cover glass was placed on a metal plate to control its temperature.…”