Antifreeze protein dispersion in eelpouts and related fishes reveals migration and climate alteration within the last 20 Ma.

Antifreeze proteins inhibit ice growth and are crucial for the survival of supercooled fish living in icy seawater. Of the four antifreeze protein types found in fishes, the globular type III from eelpouts is the one restricted to a single infraorder (Zoarcales), which is the only clade know to have antifreeze protein-producing species at both poles. Our analysis of over 60 unique antifreeze protein gene sequences from several Zoarcales species indicates this gene family arose around 18 Ma ago, in the Northern Hemisphere, supporting recent data suggesting that the Arctic Seas were ice-laden earlier than originally thought. The Antarctic was subject to widespread glaciation over 30 Ma and the Notothenioid fishes that produce an unrelated antifreeze glycoprotein extensively exploited the adjoining seas. We show that species from one Zoarcales family only encroached on this niche in the last few Ma, entering an environment already dominated by ice-resistant fishes, long after the onset of glaciation. As eelpouts are one of the dominant benthic fish groups of the deep ocean, they likely migrated from the north to Antarctica via the cold depths, losing all but the fully active isoform gene along the way. In contrast, northern species have retained both the fully active (QAE) and partially active (SP) isoforms for at least 15 Ma, which suggests that the combination of isoforms is functionally advantageous.

Solvation Layer of Antifreeze Proteins Analyzed with a Markov State Model.

Three structurally very different antifreeze proteins (AFPs) are studied, addressing the question as to what extent the hypothesized preordering-binding mechanism is still relevant in the second solvation layer of the protein and beyond. Assuming a two-state model of water, the solvation layers are analyzed with the help of molecular dynamics simulations together with a Markov state model, which investigates the local tedrahedrality of the water hydrogen-bond network around a given water molecule. It has been shown previously that this analysis can discriminate the high-entropy, high-density state of the liquid (HDL) from its more structured low-density state (LDL). All investigated proteins, regardless of whether they are an AFP or not, have a tendency to increase the amount of HDL in their second solvation layer. The ice binding site (IBS) of the antifreeze proteins counteracts that trend, with either a hole in the HDL layer or a true excess of LDL. The results correlate to a certain extent with recent experiments, which have observed ice-like vibrational (VSFG) spectra for the water atop the IBS of only a subset of antifreeze proteins. It is concluded that the preordering-binding mechanism indeed seems to play a role but is only part of the overall picture.

Electrophoresis in ice surface grooves for probing protein affinity to a specific plane of ice crystal.

Channel-like grooves are formed on the surface of frozen aqueous sucrose. They are filled with a freeze concentrated solution (FCS) and act as an efficient size-tunable separation field for micro and nanoparticles. The width of the channel can be easily varied by changing the temperature. Because the channel width decreases with decreasing temperature, particles become immobilized due to physical interference from the ice wall when the temperature reaches a threshold point specific to the particle size. Surface modification of particles can add a factor of chemical interaction between the particles and ice walls. In this study, anti-freeze proteins (AFPs) are anchored on 1µm-polystyrene (PS) particles, and their behavior in the surface grooves on the ice is studied. The threshold temperature is an effective criterion for evaluating chemical interactions between particles and ice walls. The AFP binding on 1µm PS particles lowers the threshold temperature by 2.5°C, indicating interactions between AFPs on the PS particles and the ice wall. Because the AFPs studied here show selectivity towards the prism plane, it is critical that the prism plane of the ice crystal is in contact with the FCS in the surface grooves.

RAFP-Pred: Robust Prediction of Antifreeze Proteins Using Localized Analysis of n-Peptide Compositions.

In extreme cold weather, living organisms produce Antifreeze Proteins (AFPs) to counter the otherwise lethal intracellular formation of ice. Structures and sequences of various AFPs exhibit a high degree of heterogeneity, consequently the prediction of the AFPs is considered to be a challenging task. In this research, we propose to handle this arduous manifold learning task using the notion of localized processing. In particular, an AFP sequence is segmented into two sub-segments each of which is analyzed for amino acid and di-peptide compositions. We propose to use only the most significant features using the concept of information gain (IG) followed by a random forest classification approach. The proposed RAFP-Pred achieved an excellent performance on a number of standard datasets. We report a high Youden's index (sensitivity+specificity-1) value of 0.75 on the standard independent test data set outperforming the AFP-PseAAC, AFP_PSSM, AFP-Pred, and iAFP by a margin of 0.05, 0.06, 0.14, and 0.68, respectively. The verification rate on the UniProKB dataset is found to be 83.19 percent which is substantially superior to the 57.18 percent reported for the iAFP method.

An open source cryostage and software analysis method for detection of antifreeze activity.

The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.

Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

Screening of transgenic frost-resistant cotton using a porous silicon biosensing platform.

BACKGROUND: The genetic screening of exogenous gene expression is critical for the transgenic plant breeding. OBJECTIVE: Here, a novel label-free porous silicon (PSi)-based biosensor is reported for the identification of frost-resistant cotton. METHODS: Changes in optical response signal in the presence of antifreeze protein (AFP) were detected by Fourier transform infrared (FTIR) spectromicroscopy when binding of the target AFP protein with anti-AFP-antibodies was selectively captured on the PSi biosensor. RESULTS: Compared with non-transgenic plants, significant red shifts were observed for transgenic frost-resistant cotton lines except a transgenic line 2. CONCLUSION: The approach is highly efficient with lower cost and high sensitivity for detecting large number of transgenic samples in the trial field simultaneously. This PSi biosensing platform has potential commercial applications for the rapid assessment of transgenic plants in the field.

Towards the selection of a produced water enrichment for biological gas hydrate inhibitors.

Economic concerns associated with the recovery of non-conventional hydrocarbon reserves include unexpected ice as well as ice-like gas hydrate formation. Antifreeze proteins (AFPs) inhibit ice growth, and experiments with fish, plant, and insect AFPs have shown promise of effective gas hydrate inhibition in lab-scale experiments. If produced on an industrial scale, AFPs could provide a more environmentally friendly alternative to kinetic inhibitors, but a large-scale production of these AFPs is not currently feasible. We believe that these difficulties could be surmounted by the production of microbial AFPs, but to date, only a few such proteins have been identified and purified, and none of these are associated with hydrocarbon reserves. Here, we have used ice-affinity and freeze-thaw stress to select microbes derived from oil and gas formation water, or produced water, as a source of anaerobic microbial communities. Ice-affinity successfully incorporated anaerobic bacteria under aerobic conditions, and the mixed culture had ice-associating properties. Under these conditions, ice-affinity selection does not result in cultivatable isolates, but similar, cultivable microbes were obtained following freeze-thaw selection under anaerobic conditions. Since these mixed cultures inhibited the growth of ice crystals, they also have the potential to inhibit hydrate growth. Overall, freeze-thaw selection provides a promising first step towards the isolation of microbes capable of the inhibition of ice and gas hydrate growth, for possible application for energy exploration and recovery at high-latitudes and in-deep, cold waters.

LabVIEW-operated novel nanoliter osmometer for ice binding protein investigations.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish(1), plants(2, 3), arthropods(4, 5), fungi(6), and bacteria(7). IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested(8-10), see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears(11), and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 °C), and temperature recordings as a function of time were not an available option for these instruments. We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously(9, 10), contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details. To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)(12). This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)(10). We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs(5, 13). In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations(14, 15). We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber(13). The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere(16). The Cold Stage System The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 µm in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Frozen assembly of gold nanoparticles for rapid analysis of antifreeze protein activity.

We report the novel activity-based detection of antifreeze protein (AFP), also known as ice-binding protein (IBP), using freeze-labile gold nanoparticles (AuNPs) in order to overcome labor-intensive and low throughput issues of the current method based on thermal hysteresis (TH). Upon the addition of either CnAFP from the Antarctic diatom Chaetoceros neogracile or LeIBP from the Arctic yeast Leucosporidium sp. to mercaptosuccinic acid-capped AuNP, the self-assembly of AuNPs was highly inhibited after a freezing/thawing cycle, leading to no color change in the AuNP solution. As a result, the aggregation parameter (E(520)/E(650)) of AuNP presented the rapid detection of both the concentration-dependent activity and stability of two AFPs with high sensitivity, where the detection range was 100-fold lower than that of the TH-based method. We suggest that our newly developed method is very suitable for simple and high-throughput measurement of AFP activity.

Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

Direct measurement of the thermal hysteresis of antifreeze proteins (AFPs) using sonocrystallization.

Antifreeze proteins (AFPs) are of great importance for applications in cryomedicine or the food industry. They are frequently used to lower the freezing point by preventing the growth of larger ice crystals; thus, it is paramount to determine their thermal hysteretic characteristics. However, the experimental analysis of the thermal hysteresis-an effect that is characteristic for AFPs-remains a challenging process. An easy-to-use test method for measuring the thermal hysteresis of AFPs was developed and tested with the type III AFPs. Traditional methods that have been used until now have their disadvantages and limitations. The new measurement method described in this paper allows detection of the complete cooling, freezing, heating, and melting process in a single measurement. This makes it possible to directly determine the thermal hysteresis as a functional effect of the antifreeze proteins. Measurements of the thermal hysteresis were performed by applying ultrasound to initiate the crystallization process of the antifreeze protein solution. This ultrasound technique also allows a crystallization process to be performed at defined temperature. The demonstrated results were highly reproducible and could be clearly read off the measurement curves. As a future perspective, this enables the design of automatic test devices that can be also miniaturized.

Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data.

Racemic protein crystallography offers two key features: an increased probability of crystallization and the potential advantage of phasing centric diffraction data. In this study, a phasing strategy is developed for the scenario in which a crystal is grown from a mixture in which anomalous scattering atoms have been incorporated into only one enantiomeric form of the protein molecule in an otherwise racemic mixture. The structure of a protein crystallized in such a quasi-racemic form has been determined in previous work [Pentelute et al. (2008), J. Am. Chem. Soc. 130, 9695-9701] using the multiwavelength anomalous dispersion (MAD) method. Here, it is shown that although the phases from such a crystal are not strictly centric, their approximate centricity provides a powerful way to break the phase ambiguity that ordinarily arises when using the single-wavelength anomalous dispersion (SAD) method. It is shown that good phases and electron-density maps can be obtained from a quasi-racemic protein crystal based on single-wavelength data. A prerequisite problem of how to establish the origin of the anomalous scattering substructure relative to the center of pseudo-inversion is also addressed.

Effects of cooling rate, annealing time and biological antifreeze concentration on thermal hysteresis reading.

Thermal hysteresis (TH) readings depend on the cooling rate, annealing time and the concentration of the biological antifreeze (AF) (i.e., antifreeze protein or antifreeze glycoprotein). Such time- and concentration-dependent TH readings are not true (or absolute) values. The true TH should be independent of time and AF concentration, and it should be a unique value for a given AF. Only the true TH can be used to assess the activity of an AF. A mathematical model is proposed to explain the time- and concentration-dependent behavior of AFs. The model assumes a reversible Langmuir adsorption mechanism for the AF molecules and the Kelvin effect to be applicable. A TH equation that correlates the TH reading with the cooling rate, annealing time and AF concentration was derived. The time-dependent behavior was attributed to the slow adsorption process of the AF. The theoretical calculations were compared with previously published data on the effects of the cooling rate, annealing time and AF concentration on TH readings. The calculated results agree qualitatively with the literature data. The experimental methodology required for obtaining the true TH of an AF is suggested.

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties.

Some creatures living in extremely low temperatures can produce some special materials called "antifreeze proteins" (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach "AFP-Pred" for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.

Ice-active proteins from New Zealand snow tussocks, Chionochloa macra AND C. rigida.

The ice active protein profile of New Zealand snow tussocks Chionochloa macra and C. rigida consisted of ice nucleation activity but no antifreeze or recrystallization inhibition activity. The ice nucleation activity was similar in the two species, despite them being collected at different altitudes and at different times. The activity is intrinsic to the plant and is associated with the surface of the leaves. Snow tussocks collect water from fog. Nucleation sites on the surface of their leaves may aid the efficiency of this process.

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue.

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.

Protein electrophoretic patterns and antifreezing activity in the leaf apoplast in the tropical andean species Senecio niveoaureus

Tropical high mountain plants have different adaptations to survive extreme daily temperature fluctuations and specially freezing night conditions. In winter plant species, survival to low temperatures is related to the ability of the cell to produce specific low molecular weight proteins (antifreezing proteins) and to export them to the apoplast. In order to see if high mountain tropical plants survive to low temperatures through the same mechanism we collected, during a 24 hourperiod, leaves from Senecio niveoaureus growing at 3,300 and 3,600 m.o.s.l, in the Páramo de Palacio, Chingaza, Colombia. Leaf apoplast proteins had MW between 3512 kDa. Electrophoretic patterns were different depending on the altitude and the time of sampling. However the observed variations could not be linked to changes in temperature or to the altitudinal gradient. Antifreeze activity was detected in leaf apoplast of plants at different altitudes. This is the first report of antifreeze activity in a high mountain tropical species.

Why does insect antifreeze protein from Tenebrio molitor produce pyramidal ice crystallites?

The antifreeze protein (AFP) reduces the growth rates of the ice crystal facets. In that process the ice morphology undergoes a modification. An AFP-induced surface pinning mechanism, through matching of periodic bond chains in two dimensions, enables two-dimensional regular ice-binding surfaces (IBSs) of the insect AFPs to engage a certain class of ice surfaces, called primary surfaces. They are kinetically stable surfaces with unambiguous and predetermined orientations. In this work, the orientations and molecular compositions of the primary ice surfaces that undergo growth rate reduction by the insect AFPs are obtained from first principles. Besides the basal face and primary prism, the ice surfaces engaged by insect AFPs include the specific ice pyramids produced by the insect AFP Tenebrio molitor (TmAFP). TmAFP-induced pyramids differ fundamentally from the ice pyramids produced by fish AFPs and antifreeze protein glycoproteins (AFPGs) as regards the ice surface configurations and the mode of interaction with the protein IBS. The molecular compositions of the TmAFP-induced pyramids are strongly bonded in two dimensions and have the constant face indices (101). In contrast, the molecular composition of the ice pyramids produced by fish AFPs and AFPGs are strongly bonded in only one direction and have variable face indices (h 0 l), none of which equal (101). The thus far puzzling behavior of the TmAFP in producing pyramidal crystallites is fully explained in agreement with experiment.

Systematic size study of an insect antifreeze protein and its interaction with ice.

Because of their remarkable ability to depress the freezing point of aqueous solutions, antifreeze proteins (AFPs) play a critical role in helping many organisms survive subzero temperatures. The beta-helical insect AFP structures solved to date, consisting of multiple repeating circular loops or coils, are perhaps the most regular protein structures discovered thus far. Taking an exceptional advantage of the unusually high structural regularity of insect AFPs, we have employed both semiempirical and quantum mechanics computational approaches to systematically investigate the relationship between the number of AFP coils and the AFP-ice interaction energy, an indicator of antifreeze activity. We generated a series of AFP models with varying numbers of 12-residue coils (sequence TCTxSxxCxxAx) and calculated their interaction energies with ice. Using several independent computational methods, we found that the AFP-ice interaction energy increased as the number of coils increased, until an upper bound was reached. The increase of interaction energy was significant for each of the first five coils, and there was a clear synergism that gradually diminished and even decreased with further increase of the number of coils. Our results are in excellent agreement with the recently reported experimental observations.