Molecular basis for inhibition of methane clathrate growth by a deep subsurface bacterial protein.

Methane clathrates on continental margins contain the largest stores of hydrocarbons on Earth, yet the role of biomolecules in clathrate formation and stability remains almost completely unknown. Here, we report new methane clathrate-binding proteins (CbpAs) of bacterial origin discovered in metagenomes from gas clathrate-bearing ocean sediments. CbpAs show similar suppression of methane clathrate growth as the commercial gas clathrate inhibitor polyvinylpyrrolidone and inhibit clathrate growth at lower concentrations than antifreeze proteins (AFPs) previously tested. Unlike AFPs, CbpAs are selective for clathrate over ice. CbpA3 adopts a nonglobular, extended structure with an exposed hydrophobic surface, and, unexpectedly, its TxxxAxxxAxx motif common to AFPs is buried and not involved in clathrate binding. Instead, simulations and mutagenesis suggest a bipartite interaction of CbpAs with methane clathrate, with the pyrrolidine ring of a highly conserved proline residue mediating binding by filling empty clathrate cages. The discovery that CbpAs exert such potent control on methane clathrate properties implies that biomolecules from native sediment bacteria may be important for clathrate stability and habitability.

Accumulation of Antifreeze Proteins on Ice Is Determined by Adsorption.

Antifreeze proteins (AFPs) facilitate the survival of diverse organisms in frigid environments by adsorbing to ice crystals and suppressing their growth. The rate of AFP accumulation on ice is determined by an interplay between AFP diffusion from the bulk solution to the ice-water interface and the subsequent adsorption of AFPs to the interface. To interrogate the relative importance of these two processes, here, we combine nonequilibrium fluorescence experiments with a reaction-diffusion model. We find that as diverse AFPs accumulate on ice, their concentration in the aqueous solution does not develop a gradient but remains equal to its bulk concentration throughout our experiments. These findings lead us to conclude that AFP accumulation on ice crystals, which are smaller than 100 µm in radius, is not limited by the diffusion of AFPs, but by the kinetics of AFP adsorption. Our results imply that mass transport limitations do not hinder AFPs from performing their biological function.

Micro-thermography for imaging ice crystal growth and nucleation inside non-transparent materials.

Ice crystal growth and nucleation rate measurements are usually done using light microscopy in liquid and transparent samples. Yet, the understanding of important practical problems depends on monitoring ice growth inside solid materials, for example how rapid ice growth leads to structural damage of food, or how the final structure of cementitious materials is affected by ice during curing. Imaging crystal growth inside solid materials cannot be done with visible light and is intrinsically more challenging than visible light imaging. Thermography is a technique that uses thermal (infrared) cameras to monitor temperature changes in a material, and it has been used to provide a qualitative description of ice propagation with a low spatial resolution. Here, we describe a method that uses a novel micro-thermography system to image ice nucleation and growth inside non-transparent samples. This method relies on two major components: a cold stage with accurate temperature control (±0.001 °C) and a thermal camera with high spatial and temperature resolution. Our experiments include imaging of ice formation and growth in pure water first and then inside plant leaves used as a model for a non-transparent material. An ice growth rate of 2.2 mm/s was measured inside a plant leaf at -12 °C, and ice nucleation in single plant cells was observed as a hotspot having a diameter of 160 µm. The results presented here provide an experimental proof that high-quality imaging of ice growth is achievable, thus paving the way for quantitative measurements of ice growth kinetics and ice nucleation in solid materials.

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization.

An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.

Adsorption-Inhibition of Clathrate Hydrates by Self-Assembled Nanostructures.

The mechanism by which safranine O (SFO), an ice growth inhibitor, halts the growth of single crystal tetrahydrofuran (THF) clathrate hydrates was explored using microfluidics coupled with cold stages and fluorescence microscopy. THF hydrates grown in SFO solutions exhibited morphology changes and were shaped as truncated octahedrons or hexagons. Fluorescence microscopy and microfluidics demonstrated that SFO binds to the surface of THF hydrates on specific crystal planes. Cryo-TEM experiments of aqueous solutions containing millimolar concentrations of SFO exhibited the formation of bilayered lamellae with an average thickness of 4.2±0.2 nm covering several µm2 . Altogether, these results indicate that SFO forms supramolecular lamellae in solution, which might bind to the surface of the hydrate and inhibit further growth. As an ice and hydrate inhibitor, SFO may bind to the surface of these crystals via ordered water molecules near its amine and methyl groups, similar to some antifreeze proteins.

Ice Growth Acceleration by Antifreeze Proteins Leads to Higher Thermal Hysteresis Activity.

Since some antifreeze proteins and glycoproteins (AF(G)Ps) cannot directly bind to all ice crystal planes, they change ice crystal morphology by minimizing the area of the crystal planes to which they cannot bind until crystal growth is halted. Previous studies found that growth along the c-axis (perpendicular to the basal plane, the crystal plane to which these AF(G)Ps cannot bind) is accelerated by some AF(G)Ps, while growth of other planes is inhibited. The effects of this growth acceleration on crystal morphology and on the thermal hysteresis activity are unknown to date. Understanding these effects will elucidate the mechanism of ice growth inhibition by AF(G)Ps. Using cold stages and an infrared laser, ice growth velocities and crystal morphologies in AF(G)P solutions were measured. Three types of effects on growth velocity were found: concentration-dependent acceleration, concentration-independent acceleration, and concentration-dependent deceleration. Quantitative crystal morphology measurements in AF(G)P solutions demonstrated that the adsorption rate of the proteins to ice plays a major role in determining the morphology of the bipyramidal crystal. These results demonstrate that faster adsorption rates generate bipyramidal crystals with diminished basal surfaces at higher temperatures compared to slower adsorption rates. The acceleration of growth along the c-axis generates crystals with smaller basal surfaces at higher temperatures leading to increased growth inhibition of the entire crystal.

Crystals of Benzamide, the First Polymorphous Molecular Compound, Are Helicoidal.

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X-ray diffraction reveal a complex lamellar structure of benzamide form II needle-like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non-classical morphologies.

Synergy between Antifreeze Proteins Is Driven by Complementary Ice-Binding.

In some cold-adapted organisms, over a dozen isoforms of antifreeze (glyco)proteins or AF(G)Ps are present. Although these isoforms are structurally similar, their ability to inhibit ice growth varies significantly, and, in some fish, passive isoforms can be much more abundant than the active ones. Laboratory experiments demonstrated more than a decade ago that mixtures of AFP isoforms can exhibit synergistic enhancement of each other's activity. The mechanism of this synergy effect has remained obscure and is addressed here. Using cold-stages, microfluidics, and fluorescence microscopy, the activity of binary mixtures of structurally distinct AF(G)Ps from different fish and plant species was measured. While several mixtures exhibited enhancement, some mixtures exhibited antagonism. These latter mixtures included AF(G)Ps that bind to the same crystal planes, thereby exhibiting competition. Fluorescence microscopy experiments with a synergistic mixture of two isoform types labeled with different dyes showed they bound to different crystal planes. These results helped develop a kinetic description of the mechanism by which AF(G)Ps achieve synergy. The requirements of an active isoform include high adsorption rates, and prism plane binding, while passive isoforms usually bind to a pyramidal plane at slower rates. For synergy to occur, an active isoform first binds to the faster growing prism plane. This binding slows the advancement of the prism plane and creates more pyramidal surfaces to which a passive isoform bind. These results, in part, explain the biological observation of isoform distribution in fish, and the physical chemistry of the synergistic crystal growth inhibition by two inhibitors.

Antifreeze Glycoproteins Bind Irreversibly to Ice.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) inhibit ice growth via an adsorption-inhibition mechanism that assumes irreversible binding of AF(G)Ps to embryonic ice crystals and the inhibition of further growth. The irreversible binding of antifreeze glycoproteins (AFGPs) to ice has been questioned and remains poorly understood. Here, we used microfluidics and fluorescence microscopy to investigate the nature of the binding of small and large AFGP isoforms. We found that both AFGP isoforms bind irreversibly to ice, as evidenced by microfluidic solution exchange experiments. We measured the adsorption rate of the large AFGP isoform and found it to be 50% faster than that of AFP type III. We also found that the AFGP adsorption rate decreased by 65% in the presence of borate, a well-known inhibitor of AFGP activity. Our results demonstrate that the adsorption rate of AFGPs to ice is crucial for their ice growth inhibition capability.

Dynamics and unsteady morphologies at ice interfaces driven by D2O-H2O exchange.

The growth dynamics of D2O ice in liquid H2O in a microfluidic device were investigated between the melting points of D2O ice (3.8 °C) and H2O ice (0 °C). As the temperature was decreased at rates between 0.002 °C/s and 0.1 °C/s, the ice front advanced but retreated immediately upon cessation of cooling, regardless of the temperature. This is a consequence of the competition between diffusion of H2O into the D2O ice, which favors melting of the interface, and the driving force for growth supplied by cooling. Raman microscopy tracked H/D exchange across the solid H2O-solid D2O interface, with diffusion coefficients consistent with transport of intact H2O molecules at the D2O ice interface. At fixed temperatures below 3 °C, the D2O ice front melted continuously, but at temperatures near 0 °C a scalloped interface morphology appeared with convex and concave sections that cycled between growth and retreat. This behavior, not observed for D2O ice in contact with D2O liquid or H2O ice in contact with H2O liquid, reflects a complex set of cooperative phenomena, including H/D exchange across the solid-liquid interface, latent heat exchange, local thermal gradients, and the Gibbs-Thomson effect on the melting points of the convex and concave features.

A Supramolecular Ice Growth Inhibitor.

Safranine O, a synthetic dye, was found to inhibit growth of ice at millimolar concentrations with an activity comparable to that of highly evolved antifreeze glycoproteins. Safranine inhibits growth of ice crystals along the crystallographic a-axis, resulting in bipyramidal needles extended along the <0001> directions as well as and plane-specific thermal hysteresis (TH) activity. The interaction of safranine with ice is reversible, distinct from the previously reported behavior of antifreeze proteins. Spectroscopy and molecular dynamics indicate that safranine forms aggregates in aqueous solution at micromolar concentrations. Metadynamics simulations and aggregation theory suggested that as many as 30 safranine molecules were preorganized in stacks at the concentrations where ice growth inhibition was observed. The simulations and single-crystal X-ray structure of safranine revealed regularly spaced amino and methyl substituents in the aggregates, akin to the ice-binding site of antifreeze proteins. Collectively, these observations suggest an unusual link between supramolecular assemblies of small molecules and functional proteins.

Dendrimer-Linked Antifreeze Proteins Have Superior Activity and Thermal Recovery.

By binding to ice, antifreeze proteins (AFPs) depress the freezing point of a solution and inhibit ice recrystallization if freezing does occur. Previous work showed that the activity of an AFP was incrementally increased by fusing it to another protein. Even larger increases in activity were achieved by doubling the number of ice-binding sites by dimerization. Here, we have combined the two strategies by linking multiple outward-facing AFPs to a dendrimer to significantly increase both the size of the molecule and the number of ice-binding sites. Using a heterobifunctional cross-linker, we attached between 6 and 11 type III AFPs to a second-generation polyamidoamine (G2-PAMAM) dendrimer with 16 reactive termini. This heterogeneous sample of dendrimer-linked type III constructs showed a greater than 4-fold increase in freezing point depression over that of monomeric type III AFP. This multimerized AFP was particularly effective at ice recrystallization inhibition activity, likely because it can simultaneously bind multiple ice surfaces. Additionally, attachment to the dendrimer has afforded the AFP superior recovery from heat denaturation. Linking AFPs together via polymers can generate novel reagents for controlling ice growth and recrystallization.

When are antifreeze proteins in solution essential for ice growth inhibition?

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 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.

Plant growth hormones suppress the development of Harpophora maydis, the cause of late wilt in maize.

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by rapid wilting of maize plants before tasseling and until shortly before maturity. The pathogen is currently controlled by resistant maize cultivars, but the disease is constantly spreading to new areas. The plant's late phenological stage at which the disease appears suggests that plant hormones may be involved in the pathogenesis. This work revealed that plant growth hormones, auxin (Indole-3-acetic acid) and cytokinin (kinetin), suppress H. maydis in culture media and in a detached root assay. Kinetin, and even more auxin, caused significant suppression of fungus spore germination. Gibberellic acid did not alter colony growth rate but had a signal suppressive effect on the pathogens' spore germination. In comparison, ethylene and jasmonic acid, plant senescing and defense response regulators, had minor effects on colony growth and spore germination rate. Their associate hormone, salicylic acid, had a moderate suppressive effect on spore germination and colony growth rate, and a strong influence when combined with auxin. Despite the anti-fungal auxin success in vitro, field experiments with dimethylamine salt of  2,4-dichlorophenoxyacetic acid (that mimics the influence of auxin) failed to suppress the late wilt. The lines of evidence presented here reveal the suppressive influence of the three growth hormones studied on fungal development and are important to encourage further and more in-depth examinations of this intriguing hormonal complex regulatory and its role in the maize-H. maydis interactions.

Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

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).

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

Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules.