Effects of fatty acid from deep-sea microorganisms on lipid bilayer membrane fluidity under high pressure: comparison of branched-chain and polyunsaturated fatty acid

In this study, the effects of unsaturated and saturated branchedchain fatty acids in biomembranes of microorganisms living under high temperature and high pressure on the fluidity of biomembranes were investigated by time-resolved fluorescence anisotropy measurements. First, the relationship between the order parameter S and the rotational diffusion coefficient Dw, which can be calculated from the fluorescence anisotropy measurements, and the motion of lipid molecules was investigated using lipids with three different structures, and it was found that the former was related to the spacing of lipid molecules and the latter to the motion of lipid molecules. Next, we investigated the S and Dw values of lipid bilayer membrane containing the saturated branched-chain fatty acid 12methyltridecanoic acid (12-MTA) and the polyunsaturated fatty acid (PUFA) cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). The results showed that 12-MTA increased the S value and decreased the Dw value. On the other hand, DHA tended to reduce the S value and increase the Dw value, albeit slightly. These results mean that 12-MTA narrows the molecular spacing of lipids and inhibits lipid molecular movement, while DHA tends to widen the molecular spacing of lipids and promote lipid molecular movement.


Introduction
An extreme environment, such as the deep sea or hydrothermal vents, is called an extreme environment, and the organisms that live in it are called extreme environment organisms. Extreme environment organisms adapt their cellular components to survive in such harsh environments. Among the cell members, the biological membrane is the one that is exposed to changes in the surrounding physical environment. The expression of proteins in the biomembrane is essential for maintaining the organism's life. The movement of lipid molecules around the proteins, i.e., the fluidity of the biomembrane, is necessary to make this possible. For example, organisms contain a large amount of polyunsaturated fatty acids (PUFA) in their biomembrane to prevent lipid molecules' movement from deteriorating under low temperature and high-pressure conditions such as in the deep sea. In this way, organisms are said to adjust the fatty acid composition of their biomembrane according to the surrounding environment to maintain the fluidity of the biomembrane, which is important for life support. Such a mechanism is called "homeoviscous adaptation" [1].
In addition to these reports on the temperature of Homeoviscous adaptation [2,3], adaptation to high-pressure environments has also been actively studied [4][5][6][7]. Recently, many organisms living under high pressure, such as in the deep sea, have been isolated. Their biological membranes have been shown to contain PUFAs and branched-chain fatty acids [8,9]. Although unsaturated fatty acids such as PUFA are said to increase biomembrane fluidity under low temperature, and high pressure, the effect of branched-chain fatty acids on biomembrane fluidity has not been investigated in detail. On the other hand, Gram-positive bacteria, such as Bacillus subtilis, are known to be rich in branched-chain fatty acids [10,11]. Since the tolerance of these bacteria at growth temperatures is wider on the high-temperature side, branched-chain fatty acids are thought to play some role in high-temperature environments. Although environmental conditions differ between deep-sea and hightemperature environments, branched-chain fatty acids are essential components of biological membranes for microorganisms living in these environments. Therefore, clarifying the role of branched-chain fatty acids in biological membranes may play a role in elucidating the survival strategies of organisms.
So far, research on the biological membrane fluidity of living organisms has been investigated by measuring light transmittance and differential scanning calorimetry [12,13]. However, such a measuring method regards the fluidity of the biological membrane as a change in a uniform phase state. It is difficult to observe it as the movement of lipid molecules. A technique for studying the properties of lipids at the molecular level is fluorescence anisotropy measurement. This is done by using fluorescent molecules to understand the movement of surrounding molecules. Using this technique, Usui et al. have investigated in detail the biomembrane properties of Shewanella violacea at an optimum pressure of 30 MPa [14]. As a result, they reported that EPA also has a role in preventing hyperfluidization of biomembranes, which is a new finding that differs from the general concept based on previous studies on the properties of biomembranes.
This study used time-resolved fluorescence anisotropy to investigate the detailed role of branched-chain fatty acids on biomembrane properties in a high-pressure environment. The results were compared with PUFA, which are known to enhance biomembrane fluidity in high-pressure environments.

Time-resolved fluorescence anisotropy measurements
PCI-200-PH cell holder (Shin Corporation, Kyoto) was installed on a FluoroMax-4 Spectrofluorometer (HORIBA, Ltd., Kyoto) for measurement. The measurement was performed at an excitation wavelength of λ ex = 370 nm and a fluorescence wavelength of λ em = 430 nm under a constant 70 °C and a pressure range of 0.1-15 MPa or a continuous 50 °C and a pressure range of 0.1-150 MPa. The temperature and pressure of the experimental conditions in this study were determined from the pressure-temperature phase diagram [16] of each lipid in the range that includes the liquid crystalline phase with high fluidity.

Principle of time-resolved fluorescence anisotropy measurement [17]
Light is an electromagnetic wave that has the property of a transverse wave and oscillates in a plane perpendicular to the traveling direction. On the other hand, when a fluorescent probe is excited from the ground state S 0 to the first excited state S 1 , the transition moment has an intrinsic directionality determined by the molecular structure. When this transition moment and the incident light match, the probability of being excited becomes maximum, and if orthogonal, it becomes 0. This is called light selection. In addition, the emitted fluorescence also has a unique direction. If the fluorescence polarization is strong, the polarization component of the fluorescence parallel to the incident light becomes strong. The parameter representing the degree of this polarization is the fluorescence anisotropy value r, which is expressed by the following equation.
Where I is the fluorescence intensity, its first and second subscripts represent the fluorescence component for the excitation polarization, V is the vertical direction, and H is the horizontal direction. G is the correction factor of the diffraction grating. In terms of the fluidity of the lipid membrane, the smaller the value of this anisotropy, the higher the fluidity, and the larger the value of the anisotropy, the lower the fluidity.
The fluorescence lifetime of a fluorescent probe from the time it is exciting to the time it emits fluorescence is generally several tens of seconds to several nanoseconds. In timeresolved fluorescence anisotropy measurement, the fluorescence anisotropy value is measured in nanoseconds, and the time variation of the anisotropy value shown in Eq.(2) can be obtained.
In aqueous solution, the movement of the fluorescent probe is not restricted. However, in lipid bilayers such as the one used in this study, the rotational motion of the fluorescent probe is restricted, and the value of the fluorescence anisotropy is expressed in the one-component approximation of equation (3).
Where r0 is the anisotropy at t=0, r∞ is the anisotropy when completely depolarized, and  is the The S value represents the tilt of the fluorescent probe, and the Dw value represents the rotational motion of the fluorescent probe; a smaller S value indicates a more significant tilt of the fluorescent probe, and a larger Dw value indicates a larger rotational movement of the fluorescent probe.

Evaluation of order parameter S and rotational diffusion coefficient Dw for pressure change
It is known that a single lipid bilayer shows a clear phase transition, and at high pressure, the phase state becomes low fluid, like a gel phase. Since biomembrane generally remains in a highly fluid condition. We investigated the relationship between order parameter S and rotational diffusion coefficient Dw and lipid molecules under the liquid state. The fluorescence anisotropy, r, DPPC, DSPC, and DPPE with three different structures were measured at 0.1-15 MPa, and S and D w were calculated. In this pressure range, the lipid bilayers exhibit a liquid crystalline phase. Fig. 2 shows the pressure dependence of S of lipid bilayer membrane labeled TMA-DPH. The smaller the S value, the larger the slope of the fluorescent probe. The value of DPPE showed a higher than DPPC and DSPC. This indicates that the hill of fluorescent probes in lipid bilayers prepared with DPPE is smaller than fluorescent probes in lipid bilayers designed with the other two lipids. Furthermore, the S values of DPPC and DSPC were almost the same. This means that the slope of the fluorescent probe is the same for both lipids. Fig. 3 shows the pressure dependence of Dw of lipid bilayer membrane labeled TMA-DPH. The D w value indicates the speed of rotation of the fluorescent probe, and the larger the value, the faster the process of the fluorescent probe. The D w value of DSPC is more significant than that of DPPC and DPPE. In addition, the D w values of DPPC and DPPE were almost the same.    Fig. 4 shows the fluorescence anisotropy value r in the absence and presence of DHA and 12-MTA in DPPC bilayer membrane. The r-value correlates with the fluidity of the biological membrane, and the larger the r-value, the lower the fluidity. At 50 °C, which is the measurement temperature in this study, it is known that the DPPC bilayer membrane pure system changes from a liquid crystal phase with high fluidity to a gel phase with low fluidity at a pressure of about 37 MPa. This phase change from the liquid crystal phase to the gel phase is called the primary transition, and the pressure at which the major transition occurs is called the primary transition pressure. In this study's pure system of DPPC bilayers membrane, a significant change in r value was observed at about 37 MPa, confirming the phase transition. When 12-TMA, a branched-chain fatty acid, was added to the DPPC bilayer membrane, the primary transition pressure shifted to the lower pressure side. On the other hand, when DHA, a polyunsaturated fatty acid, was added to the DPPC bilayer membrane, the primary transition pressure shifted to the high-pressure side. This indicates that 12-MTA stabilizes the gel phase, and DHA stabilizes the liquid crystal phase. On the other hand, the S and D w values of DPPC bilayer membrane in the presence of DHA were shown to be almost the same as those of the pure DPPC system, but the S values tended to be slightly higher, and the D w values marginally lower. In other words, the presence of DHA in the DPPC bilayer slightly increased the slope of the fluorescent probe, although the hill was somewhat more significant, and the motion was also slightly larger.   DPPE was higher than that of the other two lipid molecules, and the slope of the fluorescent probe was smaller. This may be attributed to the fact that DPPE, which has a relatively small ethanolamine group in the hydrophilic group of the lipid, has a denser orientation and narrower spacing between lipid molecules than the other two lipids with choline groups. DPPC and DSPC have the same hydrophilic group. The choline group and the hydrophobic group differ by two carbons. Still, this difference is not considered to be enough to change the distance between lipid molecules. Therefore, there was no clear difference in the slope of the fluorescent probes, and the S values were considered to be almost the same. These results suggest that the S value is related to the distance between lipid molecules.

Effect of branched-chain fatty acid and PUFA to order parameter S and rotational diffusion coefficient Dw under high pressure
The difference in the Dw values of the three lipids can be attributed to the difference in the structure of the hydrophobic groups of the lipids: DPPC and DPPE have 16 carbons of hydrophobic groups, whereas DSPC has two more carbons. The rotation of the fluorescent probe is affected by the movement of the lipid molecules. Therefore, it can be said that the action of lipid molecules is more significant for DSPC with an enormous D w value than for other lipids. It is considered that the more substantial number of carbons in the hydrophobic group means that the intramolecular carbon-carbon movement is more significant and the effect on the surrounding lipid molecules is greater. These results suggest that the D w value is related to the mobility of lipid molecules.

Role of branched-chain fatty acids and PUFAs on biomembrane fluidity under high pressure
The effect of 12-MTA and DHA on the biomembrane fluidity under high pressure was investigated from the S and D w values. 12-MTA increased the S value and decreased the D w value. This means that 12-MTA narrows the lipid molecular spacing and decreases the lipid molecular motion. On the other hand, DHA tended to increase the lipid spacing and lipid movement, albeit slightly. These results can be attributed to the structural differences between 12-MTA and DHA. Although 12-MTA has a branched methyl group at the second carbon from the end of the hydrocarbon chain, the hydrocarbon chain structure is much more compact than DHA. Therefore, lipid molecules in lipid bilayers containing 12-MTA tend to become denser under pressure. Still, the branched methyl group of 12-MTA creates a slight bulk at the end of the hydrocarbon chain, which may inhibit the movement of the lipid molecules. The structure of DHA contains many unsaturated bonds in the hydrocarbon chain, and when it exists in a lipid bilayer membrane, it exists in a spiral form [18]. Therefore, even if lipid molecules are tightly packed due to pressure, the bulkiness of the DHA structure is thought to create some gaps between the hydrocarbon chains of the lipid bilayer, facilitating lipid molecular movement. The presence of each fatty acid in the DPPC bilayer membrane shifted the phase transition pressure of 12-MTA to the lower pressure side and that of DHA to the higher-pressure side compared to the pure DPPC bilayer membrane system. This indicates that 12-MTA stabilizes the gel phase with low biomembrane fluidity, and DHA stabilizes the liquid crystal phase with high biomembrane fluidity. Considering that biomembrane generally needs to maintain a high degree of fluidity, the present results do not indicate that saturated branched-chain fatty acids maintain or improve the fluidity of biomembrane under pressure. In contrast, DHA tended to increase biomembrane fluidity under pressure. Regarding the relationship between pressure and branched-chain fatty acids in the biomembrane of bacteria isolated from pressure-tolerant and barophilic bacteria, it has been reported that saturated branched-chain fatty acids decrease with pressure and that increasing branched-chain fatty acids contain unsaturated bonds [8]. It has also been reported that PUFA such as DHA in the biomembranes of bacteria in the deep sea increase with pressure [5], and the results of the present study are consistent with these reports. Changes in the fatty acid composition of biomembranes with changes in temperature have been investigated not only for PUFA but also for unsaturated branched-chain fatty acids, the content of which increases as the fluidity of biomembranes decreases at low temperatures [19]. Therefore, unsaturated fatty acids may be necessary for increasing the fluidity of biomembranes, which is reduced under high pressure or lowtemperature conditions.

Conclusion
This study suggests that saturated branched-chain fatty acids may be involved in the reduction of biomembrane fluidity. In other words, saturated branched-chain fatty acids do not play a role in increasing the fluidity of biomembrane in high-pressure environments. However, when biomembrane fluidity increases in environments, such as at high temperatures, they may play a role in suppressing excessive increases in biomembrane fluidity and maintaining biomembrane fluidity that is just right for preserving life.
On the other hand, unsaturated fatty acids can increase the fluidity of biological membranes under high pressure. This result indicates that unsaturated fatty acids are involved in maintaining biomembrane fluidity sufficient for life support in a high-pressure environment.

Acknowledgement
JSPS KAKENHI Grant Number JP18K11685 supported this work.