Lipids cold uneasy: preventing the crystallization of water at -263 ° C

What is the most on the planet? What is on top of our needs along with air and food? What does one bearded joke have in common with a cucumber? The answer is simple - water. This chemical compound plays a central role in many macro and micro processes: from climate change to the chemical structure of living organisms. H ₂ O possesses a number of chemical and physical properties, which are used in one way or another by scientists of different directions. Changing certain parameters leads to the emergence of new properties or the change of old ones. From an early age, many of us know that water normally boils at 100 ° C and freezes at temperatures below 0 ° C. And then the scientists decided to change it.

Today we will get acquainted with the study, in which scientists managed to create water that does not freeze even at -263 ° C. What kind of manipulations were carried out to achieve this, what new properties and characteristics did “forever” liquid water have and what is the use of this research? We will look for answers in the report of the research group. Go.
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The basis of the study

The basis of this work is the process of preventing the crystallization of water at low temperatures. For this, it is necessary to change the geometry of the water, in which the so-called “confinement” can help, that is, retention. This mechanism prevents low-temperature crystallization of molecules into a hexagonal structure, resulting in amorphous water. Such physical retention of water at the nanometer level, scientists have called nanoconfinement. It is easier to say than to do, but scientists would not be scientists if they did not possess stubbornness and a pair of aces in the sleeve. Artificial lipids with cyclopropyl modifications in hydrophobic chains, which demonstrate a unique liquid crystal behavior at low temperature, were used as aces. These lipids allow you to maintain the amorphous state of water up to -263 ° C.

As a model of closed water in the cellular environment, scientists have chosen confinement inside soft interfaces, such as those formed during the self-organization of surface-active substances in the aquatic environment. Such a model can help to understand the mechanisms of cell survival at low temperatures.

Researchers draw our attention to the fact that size effects manifest themselves in different phases formed by hydrated monoacylglycerols * at different temperatures and levels of hydration * .

Monoacylglycerols * is a class of glycerides that consist of a glycerol molecule bound to a fatty acid via an ester (ester) bond.

Hydration * - addition to molecules or ions of water molecules.

Monoacylglycerols have a polymorphism, that is, a different crystalline structure depending on the conditions: lamellar (L _α ), inverse bicontinuous cubic (Q _II ), inverse hexagonal (H _II ), inverse micellar (L ₂ ).

The problem is that this variety of options is lost when temperatures reach room temperatures, when a general class of lipids crystallizes into a plate phase (L _c ), in which the lipid tails are packed into a long-range order crystal lattice. If the temperature drops below zero, then the coexistence of the plate-like phase L _c and ice is detected at all levels of hydration.

It turns out that you can not use these lipids? Not really. Lipids can be changed so that you can apply their positive properties, avoiding unwanted restrictions. In previous studies, scientists successfully succeeded in replacing the cis double bond in the middle of the lipid chain of monoolein with a cyclopropyl group. As a result of this manipulation, a new lipid monodihydrosterculin (MDS) will be obtained, the phase behavior of which shows the absence of a reverse hexagonal phase and the stability of the Q _II ^D phase at temperatures up to 4 ° C.

Taking as a basis the above developments and theories, the scientists presented their own research, which described a new type of lipids, forming mesophases with non-standard properties at low temperature. The most striking feature is the ability to retain glassy water at temperatures up to 10 K and at very low cooling rates.

Lipid polymorphism

To begin with, scientists explain certain nuances regarding lipid polymorphism. In nature at the moment there is a very limited number of lipids that can form Q _II phase.

Lipid chains provide the fundamental elements of all mesophases. Their molecular structure, definite length, curvature, position and degree of unsaturation together totally influence the final mesophase * .

Mesophase * - the state of matter between a liquid and a solid.

If we replace the cis-double bond of monoacylglycerols with a cis-cyclopropyl fragment, then the curvature of the chain and the length of the lipids will decrease, but the fractional compaction and lateral stress of the tails will be significantly changed. And to change the rigidity of the lipid tail, you need to change the number and position of cyclopropyl groups, as well as the length and curvature of the hydrophobic chains.

Scientists synthesized three lipids during the study (structures shown in 1a ): monodihydrosterculin (MDS), cyclopropanated lipid monolactobacillin (MLB) is an analogue of monovaccein (MV) and DCPML - monolinolein (ML).


Image number 1

The graphs above show the results of small-angle X-ray scattering (MRP): the phase diagram of the composition and temperature of the sample MLB ( 1b ), the phase diagram of the composition and temperature of the sample DCPML ( 1c ).

Judging from the observations, the hydrated MLB has a transition sequence, like in the classical monoacylglycerols ( 1b ), in which L _α , Q _II G and Q _II ^D are observed with an increase in the level of hydration. In contrast to MDS, the H _II phase is present in the MLB at high temperature.

It was possible to find out that the H _II phase and the Q _II ^D cubic phase remain stable in excess of water. This observation made it possible to determine the boundary degree of hydration for both phases by analyzing the lattice parameters at each level of hydration.

In the case of DCPML lipid, scientists observed an unusual phenomenon — the formation of the cubic phase of Q _II ^G at 22 ° C with a water content of only 5% ( 1 s ).

Previous studies have shown that the formation of H _{II by} pure hydrated monoacylglycerols is possible only at high temperatures (above room). Stable phase _II H at room and physiological temperatures (≈ 36.6 ° C) require the use of hydrophobic molecules or the presence of an ether, rather than an ester bond.

The formation of phase H _II at room temperature implies a shift of the DCPML phase diagram to lower temperatures and hydration, which was confirmed in this study.

A sample of DCPML with 12.5% ​​water was first gradually cooled to -20 ° C, and then reheated to 22 ° C. At the end of each stage of cooling and heating, the system was balanced, MPP data were also collected ( 2a ).


Image number 2

The phase transition from L _α to Q _II ^G occurs in the temperature range of –15 ... –10 ° C during the procedures of both heating and cooling. The formation of a new stable lipid cubic phase at negative temperatures was also revealed. When heated, the radius of the water channel of the Q _II ^G phase decreases from 8.4 Å at -10 ° C to 7.8 Å at 22 ° C.

As a result, scientists obtained an absolutely stable cubic phase of Q _II ^G at sub-zero temperatures. Such an observation contradicts the generally accepted facts that lipids (for example, monoolein) form cubic phases that crystallize into a plate-like crystalline phase and ice at temperatures below 0 ° C.

Properties and behavior of water

The liquid crystal nature of DCPML at negative temperatures indicates non-standard characteristics of the water contained in the nanochannels. The size of the water areas (plates or channels) can be manipulated by changing the water / lipid ratio. Melting transitions were studied using differential scanning calorimetry (DSC) measurements of mesophases at different levels of hydration ( 2b ).

DCPML samples were subjected to cyclic heat treatment (heating — cooling — heating) from −70 ° C to 60 ° C with a scanning speed of 5 ° C per minute. What we see in graph 2b was obtained during the second heating process. When the concentration of water in the sample is 20 and 25%, the peak of ice melting at 0 ° C is visible, which is typical of pure water (without added lipids). If hydration increases, this peak begins to decrease (15% of water), and then disappears completely (5% and 10% of water). The conclusion is quite obvious - confinement in the phases L _α and Q _II ^G with a low level of hydration prevents the crystallization of water at the cooling rate in question.

Also on the graph 2b you can notice small peaks at high temperatures, which correspond to transitions between different geometries and correspond to the results of MPP ( 1c ). Differences in the transition temperature by several degrees can be explained by different heating rates and, accordingly, different balancing times. Of course, do not forget about the error (1.5%), depending on the composition of different samples.

Scientists note that ice formation is present in ML at temperatures down to -60 ° C, while the amorphous state remains in DCPML. This suggests that confinement alone cannot prevent crystallization, but works in conjunction with the liquid crystal behavior of lipids to achieve this.

Next, the samples were cooled to -263 ° C at a rate of 0.1 ° C per minute, equilibrated and then heated at the same speed. In Figures 2c and 2d, we see the results of FWS measurements during heating, which show the absence of a first order transition in DCPML with low water content. Scientists have chosen a sample with a water content of 7.5% in order to provide a uniform geometry over the entire temperature range below zero.

The FWS profiles in graphs 2c and 2d do not show any jumps in the region of 0 ° C, although an increase in mobility is observed at a temperature of about -50 ° C. Scientists note that a mesophase obtained from commercial ML instead of DCPML with the same topology and water content shows melting at a temperature of about -10 ° C (peaks on the inserts at 2c and 2d ). DCPML at 15% water in the sample also shows a jump, which corresponds to the melting of ice at a temperature of about -10 ° C. However, judging by the DSC data, the transition intensity in this case is much lower, that is, only a part of the water participates in the formation of ice. And the absence of a jump for the lipid-lipid transition confirms the absence of the crystalline phase Lc in DCPML.

Experiments using wide-angle X-ray diffraction (WAXS) at low temperatures showed the hexagonal structure of ice in samples with 20% and 25 ( 2e ) hydration, as well as the absence of crystallization in the WAXS region for other samples. These observations confirm once again the liquid crystal nature of the lamellar phase (L _α ) and the absence of crystalline ice with low hydration.

Finally, scientists also used NMR spectroscopy to study the water mobility and phase behavior ( 2f ). For a sample with 7.5% water, the detection limit was reached at 0 ° C, which indicates a diffusion coefficient of less than 10 ^-11 m ² / s. And for the sample with 10% diffusion was observed to -11 ° C.

Thus, the quasilinear dependence of diffusion on temperature confirms the liquid state of water in the considered temperature range, and additional information obtained from FWS and DSC analyzes confirms the transition of water from a liquid to a glassy state at low temperatures.


Image number 3

Scientists have combined all the data collected and were able to draw up a phase diagram of the water contained in the DCPML mesophases ( 3a ).

It is worth noting that the observed processes and characteristics are closely related to the features that distinguish DCPML from all other known monoacylglycerols, namely the general shift of phase transitions towards lower temperatures and hydration, as well as the absence of L _c even at extremely low temperatures.

Image 3b shows the results of MRP lipid geometry measurements superimposed over the phase diagram of water ( 3a ). During hydration, the reverse transition L _α → Q _II ^G → L _α is observed in the temperature range from -10 ° C to 0 ° C. It is interesting that the presence of liquid water at sub-zero temperatures is associated with the stability of the cubic phase Q _II ^G. And with a decrease in hydration during cooling, a combination of lipid disorder and the geometric limitation of the L _α phase prevents the formation of ice at any temperature.

If the degree of hydration is increased, the formation of hexagonal ice will be observed. Observations showed that when hydrating 20% ​​and cooling the sample to -30 ° C, the Q _II ^G phase is stable for several hours, and no ice is detected. The transition to the L _α phase occurs after the sample is incubated for 1 hour in a temperature mode of -40 ° C, and here ice is already forming. When heated to −40 ° C, the L _α phase remains stable up to 0 ° C. In the interval -40 ... -20 ° C, the lattice parameter α shows the expected decrease (from 39.2 Å to 38.4 Å), typical of mesophases. But already between -20 ... -10 ° C, the situation is the opposite: an increase from 38.4 Å to 39.2 Å, which is usually associated with increased hydration of the lipid bilayer.

In addition to all the observations, measurements, and various scanning techniques, scientists also used molecular dynamics modeling to confirm the results of the study.


Image number 4

The researchers are well aware that the results of such modeling strongly depend on a whole set of variables: the interaction between water molecules and lipids, the lipid-lipid transition, the threshold for the transition to a glassy state, etc. However, they claim that the results of their modeling are fully consistent with the observations.

Figure 4a shows the molecular dynamic model for the melting point of lamellar mesophase with 54.3% hydration. In the center we see the starting configuration, which is partially filled with ice (white spheres) and liquid water (blue spheres). On the left is the final configuration below the melting point. And on the right - above the melting point. The top row is a system without lipids, the bottom row is with lipids (orange spheres). Image 4b is the presentation of water enclosed in the cubic phase of Q _II ^G with hydration of 54.3% for the initial (center) and final configuration below (left) and above (right) the melting temperature. In turn, graph 4c shows the temporal evolution of water above (red line) and below (black line) the melting point.

The researchers note that with low hydration, the system follows a “standard” behavior, that is, it moves from a cubic to a lamellar structure ( 4d ). When cooled, the Q _II ^G phase goes to L _α , demonstrating a sudden decrease in water mobility ( 4e ). Less mobility means that the system needs more time to balance. In this mode, the cooling process crosses the melting line after diffusion is already difficult, that is, before the crystallization of water, as a result of which we observe glassy water.

For more detailed acquaintance with the nuances of the study I strongly recommend to look into the report of scientists .

Epilogue

Scientists are accustomed to expanding the boundaries of our world view, understanding of various processes and phenomena. Some studies become the starting point of future technologies and new discoveries, and some - just food for curiosity. Today’s belongs to the first category. Understanding the behavior of the two most important elements of life (water and lipids) at extremely low temperatures can help in the development of new methods for the diagnosis and analysis of biomaterials that are difficult or even impossible to analyze at room temperatures due to their instability. Scientists also talk about the prospect of changing living cells, that is, modifying them to normal functioning under conditions of very low temperatures. In other words, if hypopsychroplanets (−50 ° C and below) and psychoplanets (−50 to 0 ° C) are considered as possible options for colonization, this study is a small step towards this.

Thank you for your attention, stay curious and have a good working week, guys!

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