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When are antifreeze proteins in solution essential for ice growth inhibition? Ran Drori, Peter L. Davies, and Ido Braslavsky Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00345 • Publication Date (Web): 06 May 2015 Downloaded from http://pubs.acs.org on May 13, 2015
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When are antifreeze proteins in solution essential for ice growth inhibition? Ran Drori1, Peter L. Davies2, Ido Braslavsky1 1
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot,
Israel, 2Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada. KEYWORDS. Antifreeze proteins, ice-binding proteins, adsorption rate, ice growth, microfluidics, basal plane, ice crystal.
Abstract Antifreeze proteins (AFPs) are a widespread class of proteins that bind to ice and facilitate the survival of organisms under freezing conditions. AFPs have enormous potential in applications that require control over ice growth. However, the nature of the binding interaction between AFPs and ice remains the subject of debate. Using a microfluidics system developed in-house we previously showed that hyperactive AFP from the Tenebrio molitor beetle, TmAFP, remains bound to an ice crystal surface after exchanging the solution surrounding the ice crystal to an AFP-free solution. Furthermore, these surface-adsorbed TmAFP molecules sufficed to prevent ice growth. These experiments provided compelling evidence for the irreversible binding of hyperactive AFPs to ice. Here, we tested a moderately active type III AFP (AFPIII) from a fish in a similar microfluidics system. We found, in solution exchange experiments, that the AFPIIIs were also irreversibly bound to the ice crystals. However, some crystals displayed “burst” growth during the solution exchange. AFPIII, like other moderately active fish AFPs, is unable to bind to the basal plane of an ice crystal. We showed that although moderate AFPs bound to ice irreversibly, moderate AFPs in solution were needed to inhibit ice growth from the bipyramidal crystal tips. Instead of binding to the basal plane, these AFPs minimized the basal face size by stabilizing other crystal planes that converge to form the crystal tips. Furthermore, when access of solution to the basal plane was physically blocked by the microfluidics device walls, we
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observed enhancement of the antifreeze activity. These findings provide direct evidence that the weak point of ice growth inhibition by fish AFPs is the basal plane, whereas insect AFPs, which can bind to the basal plane, are able to inhibit its growth and thereby increase antifreeze activity.
Introduction Terrestrial insects that survive at temperatures below –20 °C produce hyperactive antifreeze proteins (AFPs) that can depress the freezing temperature of an ice crystal in laboratory experiments by ~6° C or more 1. Marine fishes (teleosts), on the other hand, require a water freezing point depression of only ~1°C in addition to the colligative freezing point depression by their body fluids to prevent them from freezing in icy seawater
2, 3
at approximately -1.9 °C .
AFPs from fish (moderately active or moderate AFPs) are less potent than insect AFPs, and produce a freezing point depression of ~1.5°C or less 4. Depressing the freezing point of water with an AFP in the presence of ice, results in a separation between the melting and freezing temperatures. This separation is referred to as the thermal hysteresis gap (TH) and is used to characterize and quantify the AFP activity 5. The differences between the ice growth inhibition mechanisms of insect (hyperactive) and fish (moderate) AFPs are not yet fully understood. Models that can explain the adsorption of AFPs to ice have likewise not yet integrated the different attributes of the various AFPs
6, 7
. We maintain that the key difference between
hyperactive and moderate AFPs is the ability of the former to bind to the basal ice face 4, 8. Basal plane binding affects the dynamics of an AFP, such as the time-dependence of the hysteresis 8-10 crystal shaping 8, 11, 12, and ultimately the absolute TH activity 4, 8. In a previous report 13, we showed that a hyperactive AFP from the common yellow mealworm (Tenebrio molitor - TmAFP) remains bound to an ice surface after removal of the surrounding AFP solution. Fluorescence tagging indicated that this insect protein irreversibly bound to ice on many low index crystal faces including basal face. We additionally found that even when bathing AFP solution concentrations approached zero, the TH activity of these AFP-bound crystals was close to that measured at high AFP solution concentrations. The conclusion of these previous results was that once the insect AFP is bound to the ice surface, proteins in the solution are not needed to further inhibit the growth of the ice crystal. Moderate AFPs bind to prism and/or pyramidal faces of an ice crystal and limit crystal growth without binding to the basal plane
8, 14, 15
. These effects result in the growth of a bipyramidal
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crystal
11, 12
. The bipyramidal shape is characterized by reduced basal face areas, which are
localized to the two sharp tips of the bipyramidal crystal
16
, where step nucleation at this face
becomes very slow 17. Knight and DeVries calculated that a layer with a radius of 6 µm should grow under supercooled conditions below –0.1°C; however, these researchers could not measure distances on the micron scale using their system. Here, microfluidic devices observed by fluorescence microscopy were used to directly test the hypothesis of Knight and DeVries. Knight and DeVries also argued that the adsorption rates of AFPs to ice are a critical determinant of ice growth inhibition
17
. The adsorption rate is concentration-dependent;
therefore, the removal of moderate AFPs from the vicinity of a bipyramidal crystal might be expected to induce ice growth once the AFP concentration in the solution falls below the threshold needed to inhibit ice growth. As we argued earlier , the thermal hysteresis activity of hyperactive AFPs was not influence by the AFP concentration in solution after solution exchange 13. The question remains: is this also true for moderate AFPs. The kinetics of AFP adsorption onto an ice crystal surface have been studied by others
6, 7, 9
.
Sander and Tkachenko suggested a kinetic pinning model to describe how AFPs inhibit ice growth. In this model AFPs slow the advancing ice front until ice growth completely halts; whereupon AFPs accumulate on the ice surface 7. Takamichi et al. found that longer exposure times of ice crystals to AFPIII solutions increase the TH activity up to 2.5-fold 9. We previously found that the TH activity of hyperactive AFPs can be increased up to 40-fold by extending the exposure time of the crystal before cooling it further 8. We also found
8
that moderate AFPs
adsorb to the prism plane rapidly and reach saturation after a few minutes (unlike the adsorption of hyperactive AFPs to the basal plane, which reach saturation after ~4 h). Pertaya et al. used a mixture of fluorescently tagged hyperactive and moderate AFPs to obtain crystals with truncated bipyramid shapes
15
. This unusual crystal shape can be explained in terms of the kinetic
parameters of the two different AFPs. Rapid adsorption 8 and binding by the moderate AFPs to the crystal surface, followed by cooling, induced ice growth only along the c-axis. The slowly binding hyperactive AFPs then adsorbed to the basal face, and further growth along the c-axis was inhibited. Bipyramidal crystals with sharp tips could not be formed under these conditions. Clearly, further studies are needed to fully characterize the kinetic differences between moderate and hyperactive AFPs.
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In this article, we first looked at the dynamics of a moderate AFPIII (QAE1 isoform) derived from ocean pout interacting with ice. We checked whether the binding of this AFP was irreversible, as stated before photo-bleaching
19
18
and experimentally demonstrated by fluorescent recovery after
. We examined whether this fish AFP adsorbed to the ice surface sufficed to
inhibit ice growth, as does TmAFP, and the significance of the adsorption rate, and basal plane binding in ice growth inhibition. We also examined whether the TH after removal of the AFPs from the solution resembled the TH prior to solution exchange, as was observed with TmAFP.
Our report demonstrates that although the moderate AFP remained attached to the ice surfaces after reducing the AFP concentration in solution, its TH activity was not preserved. We also found that the TH of a crystal in low concentration of AFPIII was elevated when the basal plane was physically blocked to the solution by the microfluidics device walls.
Experimental section Antifreeze proteins Hyperactive TmAFP tagged with green fluorescent protein (TmAFP-GFP) was prepared as previously described
20
, and stored in a solution containing 20 mM ammonium bicarbonate
buffered at pH 8. The moderate ocean pout type III AFP (isoform QAE1) tagged with GFP (AFPIII-GFP) was also recombinantly prepared
19
, and was stored in 100 mM ammonium
bicarbonate buffered at pH 8.0.
Microfluidic settings and solution exchange experiments The microfluidic system used here has previously been described in detail 8. Briefly, a microfluidic device containing a liquid flow layer and an additional layer used to actuate the pneumatic valves was fabricated by colleagues in the Gerber lab, Bar Ilan University. The device was placed on a LabVIEW-controlled cold stage described elsewhere
13
. The cold stage was
mounted on a fluorescence microscope (Ti Eclipse, Nikon, Japan), and a sCMOS camera (Neo 5.5 sCMOS, Andor, UK) was used for video capture. A 1% BSA solution, which acted as a blocking agent to minimize the non-specific adsorption of proteins to the Polydimethylsiloxane (PDMS) surfaces of the microfluidic channel, was injected into the flow cell and incubated there for 20 min. Ammonium bicarbonate (100 mM)
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buffer (pH 8.0) was then injected to remove unbound BSA, and the temperature was lowered until ice crystal nucleation was achieved at around –20 °C. The temperature was increased towards the melting point until a ~50 µm in diameter crystal remained. A 980 nm, 500 mW, IR laser (Wuhan Laserlands Laser Equipment Co. Ltd, China) was used to melt all other ice crystals within the microfluidic channels. At that point, an AFPIII-GFP solution was injected into the flow line, and the ~50-µm crystal was melted to form a smaller crystal (15–30 µm) in the presence of the AFPIII-GFP solution. After the crystal had been coated with AFPIII-GFP molecules (by the exposure of the crystal to high AFPIII-GFP concentrations), the solution around the crystal was exchanged with ammonium bicarbonate buffer containing no AFPIIIGFP. Next, the temperature was decreased at a fixed rate (0.15 °C/min) until a crystal burst was recorded. The difference between the melting temperature of the crystal and the temperature at which this crystal ‘burst’ was defined as the TH activity.
Results AFPIII binds irreversibly to ice, but the TH activity falls after solution exchange We previously showed that TmAFP-GFP remained bound to the ice crystal surface after solution exchange, and that TmAFP-GFP molecules in the solution were not needed to inhibit ice growth 13. In the current experiment, we repeated the solution exchange procedure and compared AFPIII-GFP and TmAFP-GFP binding and activity characteristics. First, we tested the irreversible binding of AFPIII-GFP and TmAFP-GFP by measuring the fluorescence intensity of the GFP-labeled AFPs on the ice crystal surfaces during solution exchange. With both AFPs the fluorescence signal on the crystal surfaces remained constant whereas the signal in the solution decreased by two orders of magnitude (Fig. 1). Thus, binding between the AFP-GFP and the ice crystal surface was stable within the time-frame of the experiments (tested up to 30 min for AFPIII-GFP and 3 h for TmAFP-GFP). These results are in agreement with our previous findings with fluorescence recovery after photobleaching that extended to 20 h without signal recovery 19 and our previous report 13 .
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Figure 1 - Exchange of the AFP-GFP solution around ice crystals coated with AFPGFP. A and B - AFPIII-GFP, C and D - TmAFP-GFP. An ice crystal was incubated in an AFP-GFP solution (A and C), and then the solution was removed and replaced with an AFP-free buffer (B and D). The contrast in (B and D) is ~10-fold higher than in (A and C). The scale bar indicates 10 µm. E and F - The fluorescence intensities on the ice surface (black curve) and in solution (red curve) were measured for AFPIII-GFP (E) and TmAFPGFP (F). The green curve represents the calculated intensity on the ice surface after the signal from the solution had been removed, based on the equation Ice(surface)=IceSolution*Constant, where Constant is a fitting parameter that represents the solution fraction in the detection volume [13].
In the solution exchange experiment with TmAFP-GFP, the crystal was first exposed to a high concentration (20 µM) for 3 min and then the solution was exchanged. The TH activity that was measured immediately after exchange of TmAFP-GFP solution was 0.5 °C, at a residual concentration of 1 µM. Using the same experimental settings with AFPIII-GFP solutions, the TH activity measured prior to removing the solution ranged from 0.45 °C to 0.22 °C with higher AFPIII-GFP concentrations of 60 µM and 30 µM, respectively. The TH activity measured after solution exchange was much lower. Roughly 50% (7 out of 15) of the crystals examined displayed burst growth during the solution exchange process. The other half of the crystals had TH activities ranging from 0.11°C to 0.04°C at very low AFPIII-GFP concentrations (
