Average conformation of branched-chain lipid PGP-Me that accounts

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Average conformation of branched-chain lipid PGP-Me that accounts for the thermal stability and high-salinity resistance of archaeal membranes Masaki Yamagami, Hiroshi Tsuchikawa, jin cui, Yuichi Umegawa, Yusuke Miyazaki, Sangjae Seo, Wataru Shinoda, and Michio Murata Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00469 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on September 1, 2019

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Biochemistry

Average conformation of branched-chain lipid PGP-Me that accounts for the thermal stability and high-salinity resistance of archaeal membranes Masaki Yamagami†, ‡; Hiroshi Tsuchikawa†,*; Jin Cui†, ‡; Yuichi Umegawa†, ‡; Yusuke Miyazaki§; Sangjae Seo§; Wataru Shinoda§,*; Michio Murata†, ‡,* †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka

560-0043, Japan ‡

JST ERATO, Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043,

Japan §

Department of Materials Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan

KEYWORDS: archaeal lipids, enantioselective synthesis, deuterium NMR, membranes, molecular dynamics

1 ACS Paragon Plus Environment

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ABSTRACT: The average conformation of the methyl-branched chains of archaeal lipid, phosphatidyl glycerophosphate methyl ester (PGP-Me), was examined in a hydrated bilayer membrane based on the 2H NMR of enantioselectively 2Hlabeled compounds that were totally synthesized for the first time in this study. The NMR results in combination with molecular dynamics simulations revealed that the PGP-Me chain appeared to behave differently from that of typical membrane lipids such as dimyristoylphosphatidylcholine (DMPC). The C-C bonds of the PGP-Me chain adopt alternative parallel and tilted orientations to the membrane normal as opposed to a DMPC chain where all of the C-C bonds tilt in the same way on average. This characteristic orientation causes the intertwining of PGP-Me chains, which plays an important role in the excellent thermal and high-salinity stabilities of archaeal lipid bilayers and membrane proteins.

Halophytic archaea Halobacterium spp. dwell in salt-saturated environments. One of the characteristic features of these archaea is their membrane lipids, which usually possess phytanyl or methyl-branching hydrocarbon chains.1-4 One species in this genus, Halobacterium salinarium, is well known to produce bacteriorhodopsin (bR), which is a proton-pumping protein present in the purple membrane (PM).5-9 The surrounding lipids are thought to significantly contribute to the biological features of bR including its excellent thermal stability and high-salinity resistance.10-15 The bilayers of PM mainly consist of acidic polar lipids represented by phosphatidyl glycerophosphate methyl ester (PGP-Me, 1 in Figure 1).16,17 PGPMe has two characteristic structural features: an acidic bisphosphate head group and methyl-branched alkyl chains, which bind to the glycerol moiety via ether linkages in contrast to the ester linkages of usual eukaryotic lipids. Previous studies have clearly revealed that PGP-Me plays a crucial role in the structural and functional conservation of bR as a proton pump in reconstituted membranes.18,19 In addition, PGP-Me is known to significantly contribute to high-salinity resistance of PM.20 However, questions as to how and which parts of PGP-Me contribute to the functional and physical stability of PM have not yet been answered. Apart from archaeal products, membrane lipids bearing methyl-branched chains attract ample attention because of their unique features such as a lack of phase transition across a wide temperature range, thus motivating physicochemical studies by experimental21-24 and simulation methods.25-28 However, their rapid and anisotropic movement has hampered their conformational analysis in membrane. In typical mammalian phospholipids such as phosphatidylcholine and sphingomyelin, their hydrophobic moieties consisting of straight (non-substituted) alkyl chains adopt the average orientation parallel to the membrane normal.29,30 Because these chains undergo conformational change very rapidly, the conformation of chain segments cannot be determined experimentally and is usually deduced from molecular dynamics (MD) calculations. The deuterium order


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Biochemistry

parameter (SCD) obtained from 2H NMR is used as a criterion for judging whether MD simulation correctly reproduces the structural flexibility of the alkyl chain. Thus, SCD is considered to be the most reliable parameter for examining the conformation and orientation changes of the lipid molecules. SCD for the typical straight chains can further be divided into two parameters, the average orientation and the wobbling magnitude of C-D bonds (referred as a molecular order parameter). Considering this robust connection between experiment and calculation, it is of crucial importance to experimentally determine the average orientation of the methyl-branched chains, which has not been reported for the whole chain so far. 2H-labeling has been reported for the shallow positions of C1’, C2’ and C3’ in the branched hydrocarbon chain,31 but the 2H NMR data throughout the chain, particularly for the middle portion of the chain, have not been obtained. Moreover, the average conformation of C-D bonds in deuterated PGP-Me is essential information for further investigating the physicochemical properties of branched chain lipids based on NMR experiments and MD simulations. We previously succeeded in estimating the precise lateral position of each segment of the hydrocarbon chains that interacted with cholesterol in lipid bilayers based on the local mobility of the site-specific 2H-labeled phospholipids.32,33 These results inspired us to apply this strategy for investigating the PGP-Me bilayers, especially the methyl- branching methine groups. One of the main issues to be tackled in this study was how to prepare a number of site-specific 2H-labeled PGP-Me derivatives in a highly enantioselective manner since membrane properties are known to be greatly influenced by a slight heterogeneity in lipid constituents. We accomplished the first total synthesis of PGP-Me (1) via an iterative cross-coupling reaction of an enantiopure isoprene unit (≧99 % ee) as a key building block, which led to the preparation of three regioselectively 2H4-labeled PGP-Me derivatives 2, 3 and 4 at the C3’, C7’, and C11’ positions, respectively. More importantly, 2H NMR measurements using these highly enantiopure deuterated derivatives and the concomitant analysis by MD calculations disclosed interesting structural features of the branched chains, particularly their orientation toward the membrane normal, which may account for the distinctive temperature and salt stability of archaeal membranes.


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HNEt3 MeO O

P

O

O O

OH

O

PGP-Me (1) 3'-CD3,D-PGP-Me (2) 7'-CD3,D-PGP-Me (3) 11'-CD3,D-PGP-Me (4)

P

O

O

O

O

3' 7' 11' O sn-3 R1 R2 R3 R4 R5 R6

HO

+ MeO P O

O

BnO

R1

R2 R3 R4 R5 R6

Csp3-Csp3 cross-coupling

OR7 R O

OH

+

7: R7 = H, R8 = Bn 8: R7 = phytanyl, R8 = PMB

BnO R

9

OH

R

10

Isoprene unit: R9 = CH3, R10 = H CD3,D-isoprene unit: R9 = CD3, R10 = D

BnO 9

R

TsO R1

MgBr

R

O O OBn P H O

archaeol: R1 = R3 = R5 =CH3 R2 = R4 = R6 = H 1 3 5 CD3,D-archaeol: R or R or R = CD3 R2 or R4 or R6 = D

: R1 = R3 = R5 =CH3, R2 = R4 = R6 = H : R1= CD3, R2= D, R3= CH3, R4= H, R5= CH3, R6= H : R1= CH3, R2= H, R3= CD3, R4= D, R5= CH3, R6= H : R1= CH3, R2= H, R3= CH3, R4= H, R5= CD3, R6= D

8

O

10

R2 R3 R4 R5 R6

R9 = CH3, R10 = H R9 = CD3, R10 = D

+

BnO

Phytanyl tosylate R1 = R3 = R5 =CH3 R2 = R4 = R6 = H CD3,D-phytanyl tosylate R1 or R3 or R5= CD3 R2 or R4 or R6 = D

OTs

+

BrMg

Figure 1. Retrosynthetic analysis of PGP-Me (1) and CD3, D-PGP-Me derivatives (2, 3, 4)

■ MATERIALS AND METHODS Synthesis of PGP-Me and its site-selectively deuterated derivatives. Experimental procedures of the synthesis are provided as Supplementary Information. Sample preparation for 2H NMR measurements. CD3,D-PGP-Me derivatives (or D2-DMPC) (2.0 mg) were dissolved in MeOH/CHCl3 (1:1 v/v). After removing the solvent in vacuo for 20 h, the dried membrane film was added with 3.0 μL of 0.1M NaCl aq. and 0.5 mL of distilled water, and then vigorously vortexed to make multilamellar vesicles. The resultant lipid dispersion was freeze−thawed six times, lyophilized, and rehydrated with deuterium-depleted water (or with 5 M NaCl) to make 60% water (w/w)-containing liposomes. Then the mixture was again freeze−thawed and packed into a disposable high-resolution MAS insert (Bruker, Germany) sealed with Araldite epoxy glue (NICHIBAN CO. LTD, Japan). Solid-state 2H NMR measurement. 2H NMR spectra were collected on a JEOL (JEOL, Japan) ECA 400 spectrometer equipped with a 5 mm 2H static probe (Doty Scientific, USA) using a quadrupolar echo sequence.34 The 90° pulse width was 3.9 μs. Relaxation delay and the sweep width were 1.0 s and 140 kHz respectively. General procedure for MD simulations. MD simulations of DPPC and PGP-Me lipid bilayer systems have been conducted for 1μs. A DPPC bilayer system was composed of 128 DPPC, 4017 water molecules, and a PGP-Me bilayer system was composed of 128 PGP-Me, 9472 water, and 276 Na+ and 20 Cl— ions. The latter system was constructed to simulate the


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Biochemistry

membrane in 0.1M NaCl aqueous solution as in the given experimental condition. All the force field parameters employed in this work are presented in SI. The CHARMM36 force field35 was employed for lipid. The missing parameters around the ether moiety of PGP-Me were adopted from Shinoda et al.,36 and the partial atomic charges of the 2-propanol moiety linking the phosphate groups in PGP-Me were taken from the CHARMM General force field.37 All MD simulations have been carried out using the NAMD software version 2.12.38 Periodic boundary condition was applied. Pressure was controlled at 1 atm using the Langevin-piston method39 with the semi-isotropic coupling. Temperatures were set to 323 K and 303 K for DPPC and PGP-Me systems, respectively, using the Langevin thermostat. The cutoff of the nonbonded Lennard-Jones interaction was 1.2 nm with the LJ force switching function applied from 1.0 to 1.2 nm. The Coulomb interaction was calculated using the particle mesh Ewald (PME) method.40 All bond lengths including hydrogen atoms were constrained using the SHAKE algorithm.41 The time step size was 2 fs. Initial configurations were prepared using the CHARMM-GUI.42 After equilibrium MD simulation for 10 ns, 1μs production MD runs were carried out. Trajectory data were saved every 10 ps for analysis.

■ RESULTS AND DISCUSSION Synthesis of PGP-Me and CD3,D-PGP-Me derivatives. Many excellent studies have reported on the synthetic method of archaeal lipids encompassing acyclic and macrocyclic isoterpenoid chains.43-46 Our retrosynthetic analysis for PGP-Me 1 is shown in Figure 1. We envision that 1 could be synthesized by the coupling reaction between archaeol and chiral diphosphoric bisglycerol moiety, whose synthetic route was already established in our previous study.47 For archaeol, its methyl-branched side chains could be introduced through an etherification reaction of protected glycerol with phytanyl tosylate. Since the phytanyl moiety is a saturated isoprenoid, it would be efficiently constructed by the iterative carbon elongation via a CSP3-CSP3 cross-coupling reaction of compounds 11 and 13, which could be prepared from the isoprene unit. Additionally, we envisioned that this synthetic approach would make it easy to prepare several kinds of labeled PGP-Me derivatives, simply by changing the coupling order of non-labeled and deuterated isoprene units.


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O O

O 1) BnBr, KOH, toluene, 130oC HN O 2) PivCl, Et3N, THF, 20oC Bn 6 then 17, LiCl, rt

Tosylation

BnO

Tosylation

1)

TsO

95%

11

75% (2 steps)

ref 47 TsO 15

50% (2 steps)

O

LiAlH4 Et2O, 20oC 85%

BnO

Kambe coupling

TsCl Et3N, DMAP

OH

BnO

OTs

CH2Cl2, rt 95%

8 (≧99% ee)

9

CuCl2,

MgBr

Ph

THF, 0oC to rt 85%

Kambe coupling

2) Tosylation 10

O N

Bn 7 (≧99% ee)

55% (3 steps)

Debenzylation 1) BCl3, CH2Cl2 78oC

O

BnO

3) NaHMDS, CH3I, THF, -78oC

5

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BnO

12

MgBr

HO

3) Debenzylation

2) Debenzylation 71% (2 steps)

13

ref 47

O HO

1) Tosylation 2) Kambe coupling

HO

55% (4 steps)

O 16

14

76% (3 steps)

Na MeO O

P

O

O Na

O OH

O

P

O O

O O 1

Figure 2. Synthesis of enantiopure phytanol 14 and completion of total synthesis of PGP-Me (1)

We designed CD3,D-labeled PGP-Me at the three methylated positions as target 2H-labeled compounds because this kind of deuterated probe is promising for investigating the local conformation of methyl-branched parts in detail by analyzing two sets of splitting peaks of CD3 and D in 2H NMR. In order to gather information on the mobility and orientation of the methyl-branched chains in a depth dependent manner, three CD3,D-labeled PGP-Me derivatives at the C3’, C7’, and C11’ positions, respectively (2, 3 and 4) were designed, which would be synthesized using the same route via deuterated intermediates prepared from deuterated isoprene unit. One of the critical points for this synthetic plan is that a highly enantiopure (≧99% ee) isoprene unit is required since the final optical purity of PGP-Me must be over 95% ee, which is a criterion for general experiments in analyzing membrane properties. The synthesis of PGP-Me (1) began with the preparation of phytanol 14 (Figure 2). A highly enantiopure isoprene unit (8) was successfully prepared in four steps from commercially available γ-butyrolactone 5 via asymmetric methylation using Evans’ chiral oxazolidinone (6).48 Optical purity of the isoprene unit (8) was unambiguously determined by 1H NMR analysis based on the 2-methoxy-2-(1-phenyl)propionic (MαNP) acid ester method.49,50 After tosylation of compound 8, product 9 was subjected to the Cu-catalyzed CSP3-CSP3 cross-coupling reaction with isopentyl magnesium bromide developed by Kambe’s group.51-53 The reaction proceeded smoothly to produce C10 benzyl ether 10 with a 85% yield. After a two-step conversion into tosylate 11, the second Kambe coupling was performed using the chiral Grignard reagent 12, which was readily prepared from tosylate 9. Although these substrates were more complicated than the first ones, the coupling reaction occurred without difficulty and the subsequent deprotection of Bn ether furnished the C15 alcohol 13. After the same conversion, tosylation, third Kambe coupling, and then removal of the Bn group, afforded phytanol 22 with a 76%


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Biochemistry

yield for the three steps. Its enantiopurity was confirmed to be quite high by the good agreement of the optical rotation value with the reported value.54 After tosylation of enantiopure phytanol 14, product 15 was connected to a separately prepared glycerol moiety 747 and followed by debenzylation to afford archaeol 16. Finally, the coupling reaction with the chiral diphosphoric head group 6 under the same condition reported previously47 led to the completion of the first enantioselective total synthesis of PGP-Me (1) in 20 steps from γ-butyrolactone 5.

O BnO

1)

O

N O D D Bn 17 (93%D)

BnO D3C

D

NaHMDS, CD3I THF, 78oC

BnO D3C

2) LiAlH4, Et2O, 20 oC

Kambe coupling Debenzylation

3 steps

HO D 3C

75% (2 steps)

19

7 steps

OH

18 (≧99% ee)

64% (2 steps)

MgBr

D

D 3-CD3,D-phytanol (20)

MeO

38%

O

P

Na O O P OH O O

O

O Na

O 3'

O D 3C

D

3'-CD3,D-PGP-Me (2)

15 steps 15

MeO O

18%

P

O

O Na

Na O O P OH O O

O 7'

O D 3C

D

7'-CD3,D-PGP-Me (3)

16 steps MeO 15

16%

O

P

O

O Na

Na O O P OH O O

O O

11' D 3C

D

11'-CD3,D-PGP-Me (4)

Figure 3. Synthesis of CD3,D-PGP-Me derivatives (2, 3, 4)

Having established the efficient synthetic route of natural PGP-Me in a highly enantiospecific manner, we turned our attention to the preparation of three CD3,D-labeled PGP-Me derivatives (Figure 3). Following the established iterative method, 3’-CD3,D-labeled phytanol (20) was synthesized from enantiopure deuterated isoprene unit (18) prepared from 2H

2-labeled

imide 17. Further conversion of 20 was performed using essentially the same method as shown in Figure 2 to

successfully furnish the desired 3’-CD3,D-PGP-Me (2) with a good yield. As a result of simply changing the coupling order of deuterated isoprene unit (18), 7’-CD3,D-PGP-Me (3) and 11’-CD3,D-PGP-Me (4) were also uneventfully synthesized in the same manner (see Supplementary Information).


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2H

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NMR measurements. Generally, the effect of methyl-branching on membrane properties is known to enhance the

bilayer fluidity.55-58 Indeed, previous DSC, X-ray, and NMR studies have revealed that archaeal lipids can maintain the liquidcrystalline (Lα) phase across a wide range of temperatures.59-62 In the case of common straight-chained acyl groups, an anticonformer is greatly favored over a gauche conformer. However, a branched methyl group can markedly reduce the energy gap between the two rotational isomers due to the additional steric repulsion induced by the methyl group. In order to investigate the properties of archaeal lipids at the atomistic level, we examined the mobility and orientation of the side chains of PGP-Me using enantiopure and site-specific deuterated compounds 2, 3, and 4 in comparison with a eukaryotic lipid dimyristoylphosphatidylcholine (DMPC) bearing straight-chained acyl groups. Although DPPC has the same chain length (C16) as PGP-Me, we used DMPC (C14) for comparison because its lower phase transition temperature (Tm = 24oC, compared to DPPC’s Tm = 41oC) made it easy to observe the temperature dependency of

 values in a wide temperature range. In addition, to compare deuterium NMR results directly with 3’-labeled PGP-Me, we collected the data on the 4’ position of DMPC because it corresponds to the 3rd position from the carbonyl carbon along a freely rotatable chain. The 2H NMR spectrum of 3’-CD3,D-PGP-Me (2) in a temperature range from 0oC to 60oC shows two pairs of doublets (Figure 4a), which are derived from methine deuterium (smaller broad doublet) and CD3 (larger narrow doublet), respectively. Since both of the doublets revealed slightly asymmetric features, the xx+yy values corresponding to the quadrupolar splitting in axially symmetric systems were obtained by fitting the experimental spectra to simulated ones (Table S1 and Figure S3).63,64 Figure 4b depicts the 2H NMR spectrum of 4’,4’-D2-DMPC26 in the same temperature range, and the temperature dependence of quadrupolar splitting values of these lipids is summarized in Figure 4c. The splitting values of CD3 and D of 2 were almost constant from 0 to 60 oC so that the fluidity of the bilayers of 2 depended very little on temperature changes. However, the splitting values of DMPC showed significant temperature-dependence and disappearance of the doublet due to formation of the gel phase below 10oC (Figure 4).65 These results clearly reveal the importance of methyl branching for maintaining the fluidity over a wide range of temperatures.5


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Biochemistry

Figure 4. Thermal stability of PGP-Me as examined by 2H NMR spectra of aqueous multilamellar vesicles (MLVs) composed of (a) 3’,3’-CD3,D-PGP-Me and (b) 4’,4’-D2-DMPC. In spectra (a), the outer and inner doublets are derived from D and CD3, respectively. Since the spectra showed small asymmetry, the splitting value is shown as xx+yy, corresponding to 2 x  ⊥ , which is obtained from spectral simulation63,64 (Table S1). (c) Temperature dependence of quadrupolar splitting for 3’,3’CD3,D-PGP-Me and 4’,4’-D2-DMPC. *Since 20oC is lower than the phase transition temperature (Tm) of DMPC (Tm = 24oC), the gel phase may coexist in the bilayer preparation.

We next examined the salinity resistance of PGP-Me bilayers compared to DMPC bilayers. The PGP-Me producing archaea, H. salinarium, grows in an environment of high saline concentration. The live cells as well as the liposomes comprised of archaeol-based lipids are known to have a high salt tolerance.66-69 In order to perform 2H NMR measurements under conditions similar to the archaeal habitat with a large osmotic gradient, the liposomes were rehydrated with 5.0 M NaCl. As shown in the spectra (Figure 5), the PGP-Me membrane showed very similar spectral profiles in both the high and low salt conditions (Figure 5a) while the DMPC membrane gave rise to a large center peak under the 5.0 M NaCl condition (Figure 5b). Since the Pake doublet and the center peak in 2H NMR are derived from plainer bilayers and isotropic components including the micelle and cubic phase, respectively, the methyl-branched chain is chiefly responsible for the higher salinity tolerance of the planar bilayer structure, compared to the straight chain of DMPC. The cause of slightly larger quadrupole splitting widths of PGP-Me and DMPC bilayers at 5.0 M NaCl than those at 0.1 M NaCl is explained as follows. In general, phospholipids in lipid bilayers possess both attractive and repulsive forces due to van der Waals forces


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between side chains and Coulomb repulsion between charged phosphate groups, respectively. Under high salt conditions, the charge repulsion between the phosphate anions is attenuated by Na+ and Cl−. Therefore, the attraction between hydrophobic side chains becomes relatively strong, which suppresses the mobility of the side chain and leads to an increase in quadrupole splitting width. Conformation of the phytanyl chain of PGP-Me in membrane. To further investigate the membrane properties of PGPMe at the atomistic level, we examined the orientation of the phytanyl chain in bilayers based on the experimental SCD values of three branch positions using C3’-, C7’-, and C11’-deuterated derivatives 2, 3, and 4 (Figure 6a-c and Table 1). Since little information is available on the structural analysis of pure PGP-Me bilayers, we looked into diphytanoyl phosphatidylcholine (DPhPC) instead, which has the same C20 methyl-branched carbon chains as PGP-Me; its membrane properties are also quite similar to those of PGP-Me, including the bilayer thickness.21,25,66,69 First, following the conventional method, we compared the membrane thickness data previously obtained by X-ray diffraction with the SCD values in this and previous studies. Nagle et al. have reported the membrane thickness of DPhPC as a DH,H value to be 36.4 Å in hydrated bilayers,23 which is close to the thickness of dipalmitoylphosphatidylcholine (DPPC) bilayers, 37.8 Å;70 DPPC has straight acyl chains with the same longest length as DPhPC and PGP-Me. A slightly thinner (by 3—4%) DPhPC bilayer derived from MD simulations25 also suggests that the membrane thickness of PGP-Me bilayers is comparable with that of DPPC; the membrane thickness (DPP value) of PGP-Me obtained by MD simulation shown in the following section was around 37Å.

　　　　 Figure 5. Salinity resistance of PGP-Me bilayers as examined by 2H NMR spectra of aqueous multilamellar vesicles (MLVs) composed of (a) 3’,3’-CD3,D-PGP-Me and (b) 4’,4’-D2-DMPC at 30oC. Prior to 2H NMR measurements, the MLVs comprised of PGP-Me (or DMPC) were hydrated with 0.1 M and 5 M NaCl, respectively. Spectra with 0.1M NaCl are the same as in Figure 4.


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Biochemistry

On the other hand, the experimentally obtained SCD value of a C-D bond at the C7’ position of PGP-Me, 0.163, is significantly smaller than that of DPPC at the same position, 0.20.71 According to a common method conceived for lipid bilayers with straight chains,72 we converted the SCD value to segmental thickness at the C7’ position, which appeared to be 9% thinner than DPPC at the same position. The same conversions for the C3’ and C11’ segments of PGP-Me also gave rise to thinner segmental thickness than those of DPPC by 13% and >21%, respectively. Since these methyl-branched sites are known to show similar SCD values to the neighboring methylene groups,25 the considerable reduction in thickness of these segments should lead to a thinner bilayer membrane, probably by over 10%. These seemingly contradictory results may be explained by the deviation of the C-D bond orientation with respect to the membrane normal from a typical 90 o. This horizontal average orientation of the C-D bond derived from anti-conformation, referred to here as the ‘linear structure’ (Figure 6d), has been regarded as a common property29,73 for membrane lipids including PGP-Me and DPhPC.74 This contradiction has led us to propose another orientation named the ‘bent structure’; as shown in Fig. 6e, the main chain C-C bonds in this structure adopt alternative parallel and tilted orientations to the membrane normal on average. In this configuration, since the orientations of C-D and C-CD3 bonds are essentially unchanged in either anti- or gauche conformation, the membrane thickness should be less sensitive to increased gauche conformation of PGP-Me than that in the linear structure. Molecular dynamics simulations. Order parameters (SCD) at each methyl-branched position are calculated by MD simulation as shown in Table 1, where the MD data are in very good agreement with the experimental results. Thus, we were convinced that this simulation condition reproduced our experimental system quite well and the population of each conformer deduced from the MD data was reliable enough. We next deduced the population of three staggered conformers based on MD simulations, as shown in Figure S4. Surprisingly, the unfavorable gauche conformers, where the C1’ carbon atom is gauche to both C3’-Me and C4’ (see gauche— conformer in Figure 6e), turned out to be more dominant in the C2’C3’ rotational conformation.

Table 1. Splitting values and order parameters (SCD) of C-D and C-CD3 bonds at 30 oC and the SCD(CD3)/SCD(D) ratios of PGPMe from experimental and MD simulated results. NMR experimental Carbon Number

Splitting value (xx+yy) /kHz

Order Parameter |SCD|*

|SCD(CD3)/SCD(D)|

MD calculated

Order Parameter (SCD)

NMR experimental.

MD calculated.

MD calc. (Linear)

MD calc. (Bent)

CD3

D

CD3

D

CD3

D

3'

6.2

18.8

0.049

0.147

0.048

0.140

0.33

0.34

0.36

0.26

7'

5.4

20.8

0.042

0.163

0.044

0.170

0.26

0.26

0.17

0.26

11'

3.0

14.8

0.024

0.116

0.022

0.115

0.20

0.19

0.17

0.26

11

ACS Paragon Plus Environment

* |SCD| = (xx+yy) / 0 (0 = 127.5 kHz)

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Figure 6. 2H NMR spectra of (a) 3’,3’-CD3,D-PGP-Me; (b) 7’-CD3,D-PGP-Me; (c) 11’-CD3,D-PGP-Me at 30oC. Since spectra (a) and (b) showed small asymmetry, the splitting values were shown as xx+yy, corresponding to 2 x  ⊥ , which were obtained from spectral simulations63,64 (Table S1 and Figure S3). Schematic representations of (d) linear structure and (e) bent structure (see text for details). (d) In the linear structure, the average orientation of each C-C bond in phytanyl chains should be 145o as is usually the case with membrane lipids. (e) In the bent structure, the main chain C-C bonds adopt alternating parallel and tilted orientations on average, where anti-gauche rotation hardly affects the average orientation of a C-D or C-CD3 bond.

To further examine the orientation of the phytanyl chain (linear or bent structure), we focused on the ratios of the SCD values between the CD and CD3 groups, SCD(CD3)/SCD(D). Since the 3’, 7’, and 11’ positions are chiral, we had to determine 3 parameters, Euler angles ,  and a molecular order parameter (wobbling of the CD(CD3) segment) for orientation analysis, whereas only two experimental values, SCD(CD3) and SCD

(D),

were available. We used SCD(CD3)/SCD(D) ratios, which were

independent of the molecular order parameter but still reflected the orientation of these chiral centers. The SCD(CD3)/SCD(D) ratios at the three positions were calculated from the experimentally obtained and MD-derived SCD values (Table 1). Then, we examined whether the linear structure or the bent structure better explained these SCD(CD3)/SCD(D) results. The experimental ratio for the C7’ position was consistent with the value in the putative bent structure (0.26), but considerably


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Biochemistry

differed from the value deduced from the linear structure (0.17) (Figures. 6d and 6e). The result again implies that the assumption of the linear structure is not applicable to the phytanyl chain of PGP-Me, and the ‘bent structure’ is a more likely average conformation. However, unlike the C7’ position, the large SCD(CD3)/SCD(D) ratio for C3’ (Table 1) cannot be fitted into the bent conformation, but rather into the linear structure. Thus, we assumed that this discrepancy could be due to the presence of the other bent structure, which specifically occurred at the C3’ position but not at the C7’ segment. The bent structure, named ‘upward methyl-bent structure’, has an inverted orientation of the methyl group with respect to the membrane normal as illustrated in Figure 7a. MD simulations basically supported the presence of this orientation specifically for the 3’ position (Figure 7b). The angle distribution of the O-C1’ bond vector of the sn-3 chain (panel a) to the membrane normal showed that the distribution peaks appeared mainly around cos θ = −0.4 and −1.0 (red line in panel b); these highly populated orientations correspond the upward methyl-bent orientation: θ = 111o (cos θ = −0.36), and the usual bent orientation: θ =180 o (cos θ = −1.0) in Figure 6e. On the other hand, the population for the linear structure (θ =145o, cos θ = −0.82) is rather low in either the sn-2 or sn-3 chain (Figure 7b). The angle distribution of a C-CD3 bond to the membrane normal at the 3’ position (blue line in Figure 7c) showed that the most abundant orientation was located at around +0.4 (θ = 66o), which implied a higher population of the upward methyl-bent orientation since the dominant anti conformer in this structure should peak at +0.36 (θ = 69o). Since the SCD(CD3)/SCD(D) ratio at the C3’ position heavily depends on the ratio of the rotational conformation with respect to the C1’-C2’-C3’-C4’ dihedral angle, the preferential occurrence of the upward methyl-bent orientation, which gives rise to a large SCD(CD3)value than SCD(D) (Figure 7c), leads to a higher SCD(CD3)/SCD(D) ratio (0.33) than the typical one for the bent structure (0.26). So far, we described the probable reasons for the different SCD(CD3)/SCD(D) ratios between the C3’ and C7’ positions based on two kinds of bent structures. In contrast, the result of the C11’position, whose SCD(CD3)/SCD(D) ratio was markedly decreased to 0.20, allowed us to assume that the C11’ segment tends to take the intermediate orientation between the linear (0.17) and bent structures (0.26). This depth-dependent change in the average conformation was also supported by the MD simulations; Figure 7c-e shows the clear tendency that the population peaks of the C-CD3 orientation are shifted depending on the depth of the branched positions. Considering that the corresponding population peaks of methylene groups of DPPC stay unchanged (Figure 7f-h), the depth-dependent orientation change is evidently caused by the methyl branched structure. In particular, around the population peak of the C-CD3 orientation (blue traces in Figure 7c-e), the cos θ value clearly shifts from the positive area at the C3’ position to the negative area at the C11’ position, which may correspond to the change from the upward methyl-bent orientation (cos θ = 0.36) to the mixed orientation of the usual bent structure (cos θ = −0.31) and the linear structure (cos θ = 0 or −0.82). In addition, regarding the C-D bond (red traces in Figure 7c-e), a small increase of the population in the negative area for the C11’ position also reflected a more frequent


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occurrence of the linear structure compared to that of the C7’ position. A similar propensity was observed for the SCD(CD3)/SCD(D) ratio obtained from the NMR experiments. Thus, in addition to NMR data, these MD simulation results support that the phytanyl chain has an average conformation represented by the bent structure in the shallow and middle portions, and gradually relaxes into a linear structure toward the tail isopropyl group. 　　 Discussion. We will discuss the reason for the unusual thermal stability and high-salinity tolerance of PGP-Me based on the structural findings obtained in this study. Figures 8 and S6 show typical snapshots of the bilayers composed of PGP-Me (a) and DPPC (b) during the molecular dynamics simulations. The acyl chains of DPPC evidently. take on the ‘linear structure’ with the overall orientation parallel to the membrane normal. In contrast, the phytanyl chains of PGP-Me largely exhibit a twisted structure with kinked conformation, and thus disturb the close packing of side chains; this difference was supported by the simulation results, in which the distribution of angles between the chain vector (C1 to C15 vector) and the bilayer normal showed a significantly broader distribution for the PGP-Me membrane than for the DPPC membrane (Figure S5).

Figure 7. (a) Upward methyl-bent structure; (b) angle distribution of O-C1’ bond of each (sn-3 or sn-2) PGP-Me chain with respect to the outward-pointing normal vector to the membrane. (c)-(e) Angle distributions of the C-D and C-CD3 bonds of the sn-3 chain of PGP-Me with respect to the membrane normal at C3’, C7’ and C11’ position. (f)-(h) Angle distributions between each C-H bond of DPPC and the membrane normal at the C3, C7 and C11 positions of an sn-1 palmitoyl chain.


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Biochemistry

Moreover, as shown by the enlarged view in Figure 8a, a void created by the twisted chain pair of a PGP-Me molecule could be filled with a neighboring phytanyl chain in an intertwining manner. Therefore, this inter-chain interaction is expected to suppress the excessively disordered chain motion even at very high temperatures. Both 2H NMR experiments and MD simulations revealed that a C-D bond at the C7’ position has a relatively large SCD value compared with those of the other positions (Table 1), which implies that the intertwining structure frequently occurs in the middle part of a phytanyl chain. As is the case with the excellent thermal stability of PGPMe bilayers, the high salinity tolerance of archaeal membranes can also be accounted for by the intertwining structure. Previous leakage experiments of a fluorescent dye under a high osmotic pressure demonstrated that phytanyl lipids are much more resistant to leakage than saturated chain PC.57 Since the SCD(CD3)/SCD(D) ratios in 5M NaCl at 30 oC was 0.31, which was quite similar to the ratio in 0.1 M NaCl, the bent structure certainly occurs under high salinity conditions. As shown in Figure 5, with a large osmotic gradient, PGP-Me maintains the membrane fluidity and chain orientation similar to those at low salt concentration, whereas the majority of DMPC under this condition fails to retain a planar bilayer structure. In conclusion, we have achieved the highly enantioselective total synthesis of the archaeal phospholipid PGP-Me as well of three regioselectively 2H4-labeled PGP-Me derivatives. The 2H NMR analysis of these derivatives and the appropriate MD calculations enabled us to elucidate the depth-dependent average conformation of methyl-branched chains. These structural features certainly contribute to the stability of archaeal membranes across wide ranges of temperature and salinity. In particular, thickness of bilayers bearing the bent structure is less sensitive to the conformational changes of methyl-branched chains, which should contribute to the functional stability of membrane proteins such as bR under extreme conditions. The average conformation of PGP-Me in bilayers uncovered here may also help in the development of membrane materials with high thermal performance and/or salinity tolerance.


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Figure 8. Typical snapshots of a PGP-Me membrane (a) and DPPC membrane (b). Hydrophobic tails of lipids are drawn with thick sticks and colored by molecules. Headgroups including the ether linkage are shown in VDW spheres and the color codes are red: oxygen, cyan: carbon, and tan: phosphorus. VMD software75 was used to produce these snapshots. Additional snapshot pictures of PGP-Me and DPPC are shown in Figure S6.

■ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Pulication website at DOI: XXXX Details about the synthetic procedure of PGPMe/2H-labeled derivatives, solid-state NMR measurements, and MD simulations; spectral data for examining the optical purity of synthetic intermediates; additional data from solidstate NMR experiments; and 1H and 13C solution NMR of synthetic products (PDF)

■AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], telehone: (+81)-6-6850-5774 *E-mail: [email protected]


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Biochemistry

*E-mail: [email protected] ORCID Michio Murata: 0000-0002-1600-145X Wataru Shinoda: 0000-0002-3388-9227 Hiroshi Tsuchikawa: 0000-0003-0554-2303 Author Contributions H. T. and M. M. designed and supervised the project, particularly for synthesis of lipids and 2H NMR measurements. M. Y. mainly synthesized the natural product and its deuterated derivatives. W. S. designed and supervised molecular dynamics simulations. All authors contributed to the analysis, interpretation of data and drafting the manuscript. Notes The authors declare no competing ﬁnancial interest. Funding Financial support by Grants-in-Aid for Scientific Research on KAKENHI (S) (grant No. 16H06315), Innovative Areas "Frontier Research on Chemical Communications" (grant No. 17H06406), and in part by JST, ERATO Lipid Active Structure Project (JPMJER1005).

■ACKNOWLEDGMENT We are grateful to Drs. S. Hanashima and N. Inazumi (Osaka University) for their discussions and help with the NMR measurements. The calculations were performed using the facilities of the supercomputer center at Research Center for Computational Science, Okazaki, and the Institute for Solid State Physics, the University of Tokyo.

■ABBREVIATIONS Bn, benzyl; BnBr, benzyl bromide; DMAP, dimethylaminopyridine; DMPC, dimyristoylphosphatidylcholine; DPhPC, diphytanoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSC, differential scanning calorimetry; Et3N, triethylammine;

MNP,

methoxy-2-(1-phenyl)propionate;

MD,

molecular

dynamics;

NaHMDS,

sodium

bis(trimethylsilyl)amide); NMR, nuclear magnetic resonance; PC, purple membrane; PGP-Me, phosphatidyl


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glycerophosphate methyl ester; PivCl, pivaroyl chloride; PME, Particle mesh Ewald; SCD, deuterium order parameter; THF, tetrahydrofurane; TsCl, p-toluenesulfonyl chloride.

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Biochemistry

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