- Email: [email protected]
13C Nuclear magnetic resonance studies of the composition and fluidity of several chloroplast monogalactosyldiacylglycerols
653
Biochimica et Biophysics Aeta, 663 (1981) 0 Elsevier/North-Holland
Biomedical
653-660
Press
BBA 57763
‘“C NUCLEAR MAGNETIC RESONANCE STUDIES OF THE COMPOSITION AND FLUIDITY OF SEVERAL CHLOROPLAST MONOGALACTOSYLDIACYLGLYCEROLS
JAN M. CODDINGTON RICHARD I. WILLING
a, STANLEY a and DAVID
R. JOHNS a** D. RALPH G. BISHOP b
LESLIE
a,
a Division of Applied Organic Chemistry, CSIRO, G.P.O. Box 4331, Melbourne and b Plant Physiology Unit, Division of Food Research, CSIRO and School of Biological Sciences, Macquarie University, North Ryde, Sydney (Australia) (Received
August
25th,
1980)
Key words: Monogalactosyldiacylglyceroi composition; MOtional properties; Fluidity; (Chloropl~t)
‘3C-NMR; Relaxation time;
Summary The motional properties of four monogalactosyldiacylglycerols isolated from photosynthetic membranes, and containing different fatty acid chain lengths and degrees of unsaturation, have been determined using 13Cnuclear magnetic resonance. These properties have been compared with those of a lipid containing only saturated fatty acids. The 13Clongitudinal relaxation times (T,) of the carbon atoms of the acyl chains in [‘H,]methanol were measured as an index of the rates of motion of the lipid molecules and used to compare the relative fluidity of the acyl chains. The Ti values of the glyceryl and galactosyl carbon atoms in each monog~actosyldiacylglycerol are essentially constant, when allowance is made for concentration differences and the presence of two hydrogens on a tiethylene carbon versus one on a methine carbon. These results indicate similar rates of motion for the headgroup carbons in each lipid. However, for the acyl chains, the. T, values increase with the introduction of a double bond and increase further with additional unsaturation. This increase in
Supplementary data to this article are deposited with, and can be obtained from, ElsevierJNorthHolland Biomedical Press B.V., BBA Data Deposition, P-0. Box 1345.1000 BH Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/l68/57763/663 (1981t 653. The supplementaryinformation includes the lon~tudjo~ relaxation times for each carbon atom in each of the naturally occurring monog~actosyldiacyi~ycerols described here and also the data for the material obtained by hydrogenation of the Beta wiguris sample. * To whom correspondence should be addressed.
654
the rate of motion only occurs at carbon atoms beyond the first double bond in an acyl chain. These results differ to those reported for monolayer experiments where changes in packing characteristics are predominantly dependent on the introduction of the first double bond and then vary little between species.
Introduction The advent of Fourier transform techniques has seen the use of nuclear magnetic resonance (NMR), as a non-invasive probe of molecular structure and motion, advance rapidly. This has been especially evident in the investigations of natural biological materials where 13C-NMR has been used to elucidate the structures of molecules such as proteins, nucleotides and lipids, often in their biologically active states [l]. We have previously used this technique to measure the chemical shifts and relaxation times of the major lipid components of the chloroplast thylakoid membrane of Beta vulgaris [2-51. The thylakoid membrane of all photosynthetic cells is the site of the light-harvesting reactions of photosynthesis, and, with the exception of some photosynthetic bacteria, contains four characteristic lipid components; monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol and phosphatidylglycerol [ 61. Monogalactosyldiacylglycerol (Scheme I) generally comprises about 50% of the total polar lipid and in higher plants is characterized by a high content of a-linolenic acid [7]. However, in other photosynthetic cells, the fatty acid composition varies widely, not only with respect to the degree HO
HO O-~H,-~5;10R-~H,OR
R=
acyl chain
of unsaturation but also.to the length of the carbon chain (Table I). Our previous studies have shown that in free fatty acids, due to segmental motion, the longitudinal relaxation time (T,) values of the carbon atoms increase along the chain from C-2 to the terminal methyl group carbon [2]. The T1 values, and hence rates of motion, increase further with the introduction of double bonds into the chain [2]. These trends are also observed in the esterifying acyl chains of the lipids of B. uulgaris [3,4,5]. In [*H,]methanol, the lipids exist in a monomeric form and tumble freely in solution. Interactions between the acyl chains which affect their natural mobility are also observed through changes in T1 values and the different rates of motion in the saturated and unsaturated acyl chains in sulphoquinovosyldiacylglycerol can be readily detected [ 41. In this paper we report on the relative mobilities of the acyl chains of monogalactosyldiacylglycerol isolated from four natural sources, each possessing widely differing fatty acid compositions. These results are compared with those of a sample of which the acyl chains are fully saturated. These molecules do
655
not form stable bilayers in water but the info~ation on the relative mobility of each monogala~tosyldiacyl~ycerol in [ 2H4]methanol is of interest in connection with membrane studies and here is compared to their packing characteristics as described in our monolayer studies [ 81. Materials and Methods B. uulgaris was purchased at a local market. Ulua lactuca was collected from a rock shelf in the intertidal zone at Putty Beach, New South Wales. Synechococcus sp. strain number RRIMP.NI and Anacystis nidulans were grown as previously described [ 81, Lipid extractions and p~fication of individu~ monog~actosyldiacylglycerol samples were performed as previously described [3,8]. Fatty acid composition was determined by gas chromatography of the methyl esters [9]. 200 mg B. uulguris monogalactosyldiacylglycerol was hydrogenated over Pd-C to give a sample with fully saturated acyl chains. 13C-NMR showed no doublebond resonances to be present in this sample. 13C-NMR spectra were measured on 200 mg/ml preparations of the lipids in [‘H4]methanol at 36°C. Spectra were obtained in the Fourier transform mode on a Varian CFT-20 spectrometer operating at 20 MHz for the 13C nucleus with broadband proton decoupling at 80 MHz. The spectra were measured over spectral widths of 2600 Hz using 8192 data points and chemical shifts are reported relative to internal tet~ethyls~~e. Lon~tudin~ relaxation times were determined on 200 mg/ml nondegassed samples, except for the Synechococcus sp. lipid which was approx. 80 mg/ml. The fast inversion recovery technique [3,10], using the pulse sequence (180”-r-90”-2T1) was employed. Values of r were varied to obtain at least eight points within the range -SW < S, < S, and the T1 values were derived using a programme with three parameter fitting of the exponential equation. Spectra were measured over 10004% spectral widths using 8192 data points. Two sets of measurements were made so that T, values in the ranges 0.1-2.0 s and 2.0-16.0 s, could be measured conveniently. Dilution studies were performed on B. uulguris monog~actosyldiacylglycerol by adding known increments of [‘H~]meth~ol solvent to 200 mg lipid, thereby decreasing the concentration. T1 values were then measured as described above, Results and Discussion Monogalactosyldiacylglycerol compositions and 13C chemical shifts The fatty acid compositions of the monogalactosyldiacylglycerol samples are shown in Table I. The four naturally occurring monogalactosyldiacylglycerols were selected from those used for monolayer studies [8] on the basis of fatty acid chain length and degree of unsaturation. Thus the content of the fatty acids with sixteen carbon atoms varies from 89% in A. niduluns monogalactosyldiacylglycerol to less than 15% in I?. uu~guris monog~actosyldiacylglycerol. Concomit~tly the Double-Bond Index (DBI) [8] (a weighted average of unsaturation) increases from 0.6 in A. nidulans to 3.3 in U. lactucu monogalacto-
656 TABLE
I
FATTY
ACID
THETIC
MEMBRANES
The
fatty
ent. Thus
COMPOSiTION
acids
are
described
hexadecanoic
acid
OF
here
MONOGALACTOSYLDIACYLGLYCEROL
according
is represented
to by
their
16:0
chain
and
length
(QZ,
122,
FROM
and
the number
PHOTOSYN-
of double
bonds
15Z)-octadeca-9.12.15~trienoic
pres-
acid
by
18:3 Acid
Hydrogenated
A. nidulans
Synechococcus
sp.
B. vulgaris
u. kxtuco
B. vulgaris 14:o
-
15:o
-
16:0
10.5 -
16:l 16:2
0.6
-
2.7
38.0
2.2
2.1
47.6
13.7
1.4
0.3
0.5 -
1.2
0.7
5.5 36.4
11.0 -
-
16:3 16:4
-
17:l 18:O 18:l 18:2
0.7
-
8.0
24.9
1.2
89.5 -
%Cl
bond
index
0
2.1 83.1 2.9
3.3
52.2
89.4
-
3.3 3.7
1.0
0.6
10.5
acids
6
0.3
32.7 13.7
14.4 -
18:4 Double
-
8.4
-
18:3
-
-
39.1
14.7
46.6
syldiacylglycerol. A. ~~~u~a~s monog~actosyldiacylglycerol lacks polyunsaturated fatty acids, while Synechococcus sp. monog~actosyldiacylglycerol contains 39% mono-unsaturated and 23% polyunsaturated acids. Monogalactosyldiacylglycerol from B. uulguris contains 84% trienoic acids while that from U. Zuctuca contains 38% trienoic and 50% tetraenoic acids. The major acylating acids in each monogalactosyldiacylglycerol sample were detected and assigned from the 13C-NMR spectrum *. Chemical shift assignments correspond closely in all cases to those of the appropriate carbon in the acyl chains of the free fatty acids [2]. As indicated previously [3], the only significant difference observed is a solvent shift of approx. 0.5 ppm on changing from C2HC13 to [2H~]meth~ol. Only one set of chemical shifts for the glyceryl and galactosyl carbons were observed in each monog~actosyldiacyl~ycerol sample, indicating that differences in fatty acid composition have no detectable effect on the shifts of the headgroup nuclei. The coincidence in chemical shift of the carbons in a series of saturated, mono-unsaturated and di-unsaturated acids with chain lengths between 14 and 18 carbon atoms makes it impossible to distinguish carbons within a series. The spectra of the hexadecatetraenoic (16 : 4) and octadecatetraenoic (18 : 4) acyl chains of U. lactuca are previously unreported. The r3C chemical shifts have been assigned from the substituent chemical shifts previously described [2,11] and are shown in Table II.
* Those this
acids
technique.
‘which
make
up
only
a small
fraction
of
the
total
(less
than
10%)
were
not
detected
using
657
TABLE l3C
II
CHEMICAL
SHIFTS
OF MAJOR
FATTY
ACfD
GROUPS
IN U. LACTUCA
MONOGALACTOSYL-
DIACYLGLYCEROL Carbon
16:4
18:3
18:4
2 3 4 5 6 7 8 9 10
34.8 23.6 125.9 *
34.8 25.8 30.0 30.0 30.0 30.5 28.0 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
34.8 25.4 30.0 27.7 129.1 130.4 26.4 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
11 12 13 14 15 16 17 18
26.4 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
-
* C-5 of the 16:4 acidcannotbe assignedby the methodsdescribedbutis mostlikely to be at 132.6 PPm.
13C longitudinal relaxation times Dilution studies. The relaxation time of the carbon atoms of a free fatty acid is concentration-dependent [Z] and varies with the viscosity of the medium. In dilute solutions, the T1 values are longer which is indicative of faster rates of motion. This effect is also observed with the monogalactosyldiacylglycerol samples in [2H4]methanol. A comparison of the TXvalues for B. uulguris monogalactosyldiacylglycerol at a range of concentrations (68, 100 and 200 mg/ml) shows a trend to longer values with decreasing concentration (Fig. la and b). The rate of change of T1 values generally increases towards the carbons of the methyl end of the acyl chain, but the overall trend is also apparent in the glyceryl and galactosyl carbon atoms where the relative change in T1 is small and similar to those of the first few carbon atoms of the acyl chain (Fig. lb). These dilution effects must be borne in mind when comparing the relaxation data from the different sources. Specifically, the data for Synechococcus sp. monog~ac~syldia~yl~ycerol yvere obtained using only 80 mg/ml of material and the Ti values seem anomalously long. Comparative studies. The 13C longitudinal relaxation times for the carbon atoms in the monogalactosyldiacylglycerol samples from the four organisms and the hydrogenated B. uulguris monogalactosyldiacylglycerol have been determined and the data are deposited in the BBA Data Bank. In all the monog~a~tosyldiacylglycerol molecules studied, the T1 values of the glyceryl and galactosyl carbon atoms of the headgroup remain constant (the higher values of 0.35-0.55 s for Synechococcus sp. monogaIactosyldiacylglycero1 are due to its lower concentration) with the glyceryl carbon atoms being consistently shorter. Typical values are 0.35 s for the galactosyl carbons and 0.25 s for the glyceryl carbons. The glyceryl chain is held quite rigidly and the galactosyl ring
658
A
12.13x_ 11,14x,
\
-.
. ‘\.
--x_
--__
-_
‘\
--__ ---
IOX- _ ‘x__ 9”-_-‘-.x__;-----‘r--z:==--_
-
----__ _ _--_--_=
-x12,13 ~ll.14 x9.10
MGG
g I=
05-
1
o-4-
Ti 0 a
0.3
-d c -a f t F0.l 4
o-z
Concentration
\
-
---__ 1’. \
-
(mg/ml)
\
---___
I---___ \ *______----=7--c_
._
_----*,
--
---_*3’
-
1 0
I
1
I
I
I
50
68
100
150
200
MGG
Conccntratlon
(mg/ml)
Fig. 1. Dilution effects on longitudinal relaxation times of B. vulgaris monogalactosyldiacylglycerol (MGG). (a) T1 values (in seconds) for the carbons atoms of the acyl chain vs. concentration (mg/ml). (b) T1 values (in seconds) for the headgroup glyceryl and galactosyl carbon atoms vs. concentration (mg/ml).
has only slow, restricted movement regardless of the degree of unsaturation and motions of the esterifying acyl chains. In contrast, the T1 values of the acyl chain carbons vary considerably in the five samples studied. The shortest T1 values, for the equivalent acyl chain carbon atoms are observed for the fully saturated sample with a range of values between 0.45 and 4.2 s. The introduction of a single double bond into one of the acyl chains, as in A. niduluns and Synechococcus sp. monogalactosyldiacylglycerols, increases the T1 values of the carbon atoms of the saturated chain to between 0.37 and 5.4 s. This interaction between saturated and unsaturated acyl chains has been previously noted by us in the plant lipid, sulphoquinovosyldiacylglycerol [4]. The major
659
difference in rates of motion, however, occurs in the unsaturated acyl chain at carbon atoms beyond the double bond where typically, at C-11, the relaxation times may increase from 0.9 to 2.0 s. There is further increase in the T1 values with the introduction of additional double bonds into the chain. Thus the T1 values for the acyl chains of U. Zactuca are consistently longer than those in A. nidulans, indicating that, in U. lactuca, considerably faster motion is occurring. For example, the terminal methyl group T1 value increases from 5.4 to 8.1 s. As is the case in the free fatty acids in chloroform [ 21, and the purified chloroplast lipids of B. uulguris [3--51, this increase in T1 value is related to the segmental motion along the chain. For saturated chains this requires @coupled truns-gauche rotations about C-C bonds while in unsaturated chains the more energetically likely single trunsgauche rotation about the bond adjacent to the double bond is able to induce motion. In effect, the introduction of a double bond into the acyl chain permits more freedom of movement. Polyunsaturated acyl chains should then have even more mobility and this is reflected in the T1 values which show that the rate of motion within the acyl chains of the monogalactosyldiacylglycerol samples increases markedly beyond each double bond. The effect is cumulative and as the degree of unsaturation increases so does the overall fluidity of the acyl chain. At carbon atoms before the position of the double bond, those up to C-S, the T1 values for all the acylating moieties, except for those of the 16 : 4 and the 18 : 4 acids, are very similar and occur in the range of 0.5-1.0 s, again indicating that changes in mobility are primarily induced by the double bonds present. These results on the relative mobilities of the acyl chains of the various monogalactosyldiacylglycerol samples may be compared to monolayer experiments which give information on the packing characteristics of the molecules [ 81. The monolayer behaviour of the different monogalactosyldiacylglycerol samples is not directly related to the inherent motional properties of the monomeric species studied here. The major differences between the fully saturated monogalactosyldiacylglycerol and the unsaturated samples demonstrate that packing in a monolayer is dependent upon the volume of the acyl chains rather than the headgroups and that the introduction of a single double bond is primarily responsible for changes in the packing behaviour. Further unsaturation has very little effect. The similar packing characteristics in the unsaturated samples studied may also reflect the position of the double bond since in all four natural monogalactosyldiacylglycerol samples, the double bond closest to the headgroup occurs between C-8 and C-9. For other monogalactosyldiacylglycerol molecules with their first double bond closer to the headgroup the monolayer pressure vs. area curves may be different from those reported. This dependence on the first double bond for variation of properties is also reflected in the 13C longitudinal relaxation time data where the T1 values only change significantly at carbon atoms at, and beyond, the position of this double bond. The major difference between the monolayer and relaxation time studies is that the packing characteristics depend only on the presence of a double bond, whereas the T1 values change with the degree of unsaturation. The use of the two techniques provides complementary information on the monogalactosyldiacylglycerol molecules, since we obtain knowledge of the static arrangements
660
from the monolayer studies and data on the dynamics of motion and fluidity from 13C-NMR. There is wide variation between the monogalactosyldiacylglycerols of higher plants and that of other species, in the degree of unsaturation and mobility of the acyl chains. Since monogalactosyldiacylglycerol constitutes about half of the polar lipid of the photosynthetic membranes, its behaviour is expected to typify that of the whole membrane bilayer. The range of mobilities found in the acyl chains of monogalactosyldiacylglycerol molecules for different species indicates that the fluidity of the membrane may vary within wide limits without affecting the functional photosynthetic integrity of the system, and indeed, it has been reported that partial hydrogenation of whole chloroplasts has no significant effect on photosynthetic electron transport [12]. The biological rationale for the diversity of unsaturation in monogalactosyldiacylglycerol molecules remains unclear. References Campbell,
I.D.
and
Dobson.
C.M.
(1979)
Methods
Biochem.
Anal.
25,
l-133
Johns.
S.R..
Leslie.
D.R.,
Willing.
R.I.
and
Bishop,
D.G.
(1977)
Aust.
J. Chem.
30,813-822
Johns,
S.R..
Leslie.
D.R.,
Willing,
R.I.
and
Bishop,
D.G.
(1977)
Aust.
J. Chem.
30.
823-834
Johns,
S.R.,
Leslie,
D.R.,
Willing,
R.I.
and
Bishop,
D.G.
(1978)
Aust.
J. Chem.
31.
65-72
J.M.,
Johns,
Coddington.
S.R.,
Leslie,
D.R.,
WiIIing.
R.I.
and
Bishop,
D.G.
(1981)
Aust.
J. Chem.,
in
the press 6
Bishop,
D.G.,
(Deamer. 7 8 9
ed.).
Benson,
A.A.
Springer,
Berlin
Bishop. Phys.
10
Nolan.
D.W..
Bishop, &net,
(1971)
D.G.. Acta
11
Batchelor.
12
Restall,
Bain,
Levy, J.G.,
C.J.,
555.119-130
in
Kenrick,
Johns,
269-288,
S.R.
and
Academic
Structure
and
J.R..
Bayston,
and
Downton.
Willing. Press,
Function
J.H.,
R.I.
New of
(1978)
in
Light-Transducing
Membranes
York Chloroplasts
MacPherson,
AS.
and
(Gibbs. Johns,
M., S.R.
ed.), (1980)
pp.
129-148,
Biochim.
Bio-
Biophys.
Acta
602,248-259
D.G., D.,
W.G.. pp.
J.M.
G.C.
and Peat.
Cushley,
Williams,
R.J.
I.R. and
P.. Percival,
W.J.S. (1975)
Prestegard, M.P.,
(1976)
J. Magn. J.H.
Quinn,
Aust.
(1974)
P.J.
J. Plant.
Resonance and
18.
J. Org. Chapman.
Physiol.
3. 3340
199-204
Chem.
39,
D. (1979)
1698-1705 Biochim.
Biochimica et Biophysics Aeta, 663 (1981) 0 Elsevier/North-Holland
Biomedical
653-660
Press
BBA 57763
‘“C NUCLEAR MAGNETIC RESONANCE STUDIES OF THE COMPOSITION AND FLUIDITY OF SEVERAL CHLOROPLAST MONOGALACTOSYLDIACYLGLYCEROLS
JAN M. CODDINGTON RICHARD I. WILLING
a, STANLEY a and DAVID
R. JOHNS a** D. RALPH G. BISHOP b
LESLIE
a,
a Division of Applied Organic Chemistry, CSIRO, G.P.O. Box 4331, Melbourne and b Plant Physiology Unit, Division of Food Research, CSIRO and School of Biological Sciences, Macquarie University, North Ryde, Sydney (Australia) (Received
August
25th,
1980)
Key words: Monogalactosyldiacylglyceroi composition; MOtional properties; Fluidity; (Chloropl~t)
‘3C-NMR; Relaxation time;
Summary The motional properties of four monogalactosyldiacylglycerols isolated from photosynthetic membranes, and containing different fatty acid chain lengths and degrees of unsaturation, have been determined using 13Cnuclear magnetic resonance. These properties have been compared with those of a lipid containing only saturated fatty acids. The 13Clongitudinal relaxation times (T,) of the carbon atoms of the acyl chains in [‘H,]methanol were measured as an index of the rates of motion of the lipid molecules and used to compare the relative fluidity of the acyl chains. The Ti values of the glyceryl and galactosyl carbon atoms in each monog~actosyldiacylglycerol are essentially constant, when allowance is made for concentration differences and the presence of two hydrogens on a tiethylene carbon versus one on a methine carbon. These results indicate similar rates of motion for the headgroup carbons in each lipid. However, for the acyl chains, the. T, values increase with the introduction of a double bond and increase further with additional unsaturation. This increase in
Supplementary data to this article are deposited with, and can be obtained from, ElsevierJNorthHolland Biomedical Press B.V., BBA Data Deposition, P-0. Box 1345.1000 BH Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/l68/57763/663 (1981t 653. The supplementaryinformation includes the lon~tudjo~ relaxation times for each carbon atom in each of the naturally occurring monog~actosyldiacyi~ycerols described here and also the data for the material obtained by hydrogenation of the Beta wiguris sample. * To whom correspondence should be addressed.
654
the rate of motion only occurs at carbon atoms beyond the first double bond in an acyl chain. These results differ to those reported for monolayer experiments where changes in packing characteristics are predominantly dependent on the introduction of the first double bond and then vary little between species.
Introduction The advent of Fourier transform techniques has seen the use of nuclear magnetic resonance (NMR), as a non-invasive probe of molecular structure and motion, advance rapidly. This has been especially evident in the investigations of natural biological materials where 13C-NMR has been used to elucidate the structures of molecules such as proteins, nucleotides and lipids, often in their biologically active states [l]. We have previously used this technique to measure the chemical shifts and relaxation times of the major lipid components of the chloroplast thylakoid membrane of Beta vulgaris [2-51. The thylakoid membrane of all photosynthetic cells is the site of the light-harvesting reactions of photosynthesis, and, with the exception of some photosynthetic bacteria, contains four characteristic lipid components; monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol and phosphatidylglycerol [ 61. Monogalactosyldiacylglycerol (Scheme I) generally comprises about 50% of the total polar lipid and in higher plants is characterized by a high content of a-linolenic acid [7]. However, in other photosynthetic cells, the fatty acid composition varies widely, not only with respect to the degree HO
HO O-~H,-~5;10R-~H,OR
R=
acyl chain
of unsaturation but also.to the length of the carbon chain (Table I). Our previous studies have shown that in free fatty acids, due to segmental motion, the longitudinal relaxation time (T,) values of the carbon atoms increase along the chain from C-2 to the terminal methyl group carbon [2]. The T1 values, and hence rates of motion, increase further with the introduction of double bonds into the chain [2]. These trends are also observed in the esterifying acyl chains of the lipids of B. uulgaris [3,4,5]. In [*H,]methanol, the lipids exist in a monomeric form and tumble freely in solution. Interactions between the acyl chains which affect their natural mobility are also observed through changes in T1 values and the different rates of motion in the saturated and unsaturated acyl chains in sulphoquinovosyldiacylglycerol can be readily detected [ 41. In this paper we report on the relative mobilities of the acyl chains of monogalactosyldiacylglycerol isolated from four natural sources, each possessing widely differing fatty acid compositions. These results are compared with those of a sample of which the acyl chains are fully saturated. These molecules do
655
not form stable bilayers in water but the info~ation on the relative mobility of each monogala~tosyldiacyl~ycerol in [ 2H4]methanol is of interest in connection with membrane studies and here is compared to their packing characteristics as described in our monolayer studies [ 81. Materials and Methods B. uulgaris was purchased at a local market. Ulua lactuca was collected from a rock shelf in the intertidal zone at Putty Beach, New South Wales. Synechococcus sp. strain number RRIMP.NI and Anacystis nidulans were grown as previously described [ 81, Lipid extractions and p~fication of individu~ monog~actosyldiacylglycerol samples were performed as previously described [3,8]. Fatty acid composition was determined by gas chromatography of the methyl esters [9]. 200 mg B. uulguris monogalactosyldiacylglycerol was hydrogenated over Pd-C to give a sample with fully saturated acyl chains. 13C-NMR showed no doublebond resonances to be present in this sample. 13C-NMR spectra were measured on 200 mg/ml preparations of the lipids in [‘H4]methanol at 36°C. Spectra were obtained in the Fourier transform mode on a Varian CFT-20 spectrometer operating at 20 MHz for the 13C nucleus with broadband proton decoupling at 80 MHz. The spectra were measured over spectral widths of 2600 Hz using 8192 data points and chemical shifts are reported relative to internal tet~ethyls~~e. Lon~tudin~ relaxation times were determined on 200 mg/ml nondegassed samples, except for the Synechococcus sp. lipid which was approx. 80 mg/ml. The fast inversion recovery technique [3,10], using the pulse sequence (180”-r-90”-2T1) was employed. Values of r were varied to obtain at least eight points within the range -SW < S, < S, and the T1 values were derived using a programme with three parameter fitting of the exponential equation. Spectra were measured over 10004% spectral widths using 8192 data points. Two sets of measurements were made so that T, values in the ranges 0.1-2.0 s and 2.0-16.0 s, could be measured conveniently. Dilution studies were performed on B. uulguris monog~actosyldiacylglycerol by adding known increments of [‘H~]meth~ol solvent to 200 mg lipid, thereby decreasing the concentration. T1 values were then measured as described above, Results and Discussion Monogalactosyldiacylglycerol compositions and 13C chemical shifts The fatty acid compositions of the monogalactosyldiacylglycerol samples are shown in Table I. The four naturally occurring monogalactosyldiacylglycerols were selected from those used for monolayer studies [8] on the basis of fatty acid chain length and degree of unsaturation. Thus the content of the fatty acids with sixteen carbon atoms varies from 89% in A. niduluns monogalactosyldiacylglycerol to less than 15% in I?. uu~guris monog~actosyldiacylglycerol. Concomit~tly the Double-Bond Index (DBI) [8] (a weighted average of unsaturation) increases from 0.6 in A. nidulans to 3.3 in U. lactucu monogalacto-
656 TABLE
I
FATTY
ACID
THETIC
MEMBRANES
The
fatty
ent. Thus
COMPOSiTION
acids
are
described
hexadecanoic
acid
OF
here
MONOGALACTOSYLDIACYLGLYCEROL
according
is represented
to by
their
16:0
chain
and
length
(QZ,
122,
FROM
and
the number
PHOTOSYN-
of double
bonds
15Z)-octadeca-9.12.15~trienoic
pres-
acid
by
18:3 Acid
Hydrogenated
A. nidulans
Synechococcus
sp.
B. vulgaris
u. kxtuco
B. vulgaris 14:o
-
15:o
-
16:0
10.5 -
16:l 16:2
0.6
-
2.7
38.0
2.2
2.1
47.6
13.7
1.4
0.3
0.5 -
1.2
0.7
5.5 36.4
11.0 -
-
16:3 16:4
-
17:l 18:O 18:l 18:2
0.7
-
8.0
24.9
1.2
89.5 -
%Cl
bond
index
0
2.1 83.1 2.9
3.3
52.2
89.4
-
3.3 3.7
1.0
0.6
10.5
acids
6
0.3
32.7 13.7
14.4 -
18:4 Double
-
8.4
-
18:3
-
-
39.1
14.7
46.6
syldiacylglycerol. A. ~~~u~a~s monog~actosyldiacylglycerol lacks polyunsaturated fatty acids, while Synechococcus sp. monog~actosyldiacylglycerol contains 39% mono-unsaturated and 23% polyunsaturated acids. Monogalactosyldiacylglycerol from B. uulguris contains 84% trienoic acids while that from U. Zuctuca contains 38% trienoic and 50% tetraenoic acids. The major acylating acids in each monogalactosyldiacylglycerol sample were detected and assigned from the 13C-NMR spectrum *. Chemical shift assignments correspond closely in all cases to those of the appropriate carbon in the acyl chains of the free fatty acids [2]. As indicated previously [3], the only significant difference observed is a solvent shift of approx. 0.5 ppm on changing from C2HC13 to [2H~]meth~ol. Only one set of chemical shifts for the glyceryl and galactosyl carbons were observed in each monog~actosyldiacyl~ycerol sample, indicating that differences in fatty acid composition have no detectable effect on the shifts of the headgroup nuclei. The coincidence in chemical shift of the carbons in a series of saturated, mono-unsaturated and di-unsaturated acids with chain lengths between 14 and 18 carbon atoms makes it impossible to distinguish carbons within a series. The spectra of the hexadecatetraenoic (16 : 4) and octadecatetraenoic (18 : 4) acyl chains of U. lactuca are previously unreported. The r3C chemical shifts have been assigned from the substituent chemical shifts previously described [2,11] and are shown in Table II.
* Those this
acids
technique.
‘which
make
up
only
a small
fraction
of
the
total
(less
than
10%)
were
not
detected
using
657
TABLE l3C
II
CHEMICAL
SHIFTS
OF MAJOR
FATTY
ACfD
GROUPS
IN U. LACTUCA
MONOGALACTOSYL-
DIACYLGLYCEROL Carbon
16:4
18:3
18:4
2 3 4 5 6 7 8 9 10
34.8 23.6 125.9 *
34.8 25.8 30.0 30.0 30.0 30.5 28.0 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
34.8 25.4 30.0 27.7 129.1 130.4 26.4 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
11 12 13 14 15 16 17 18
26.4 130.8 128.8 26.4 129.1 129.1 26.4 128.0 132.6 21.3 14.6
-
* C-5 of the 16:4 acidcannotbe assignedby the methodsdescribedbutis mostlikely to be at 132.6 PPm.
13C longitudinal relaxation times Dilution studies. The relaxation time of the carbon atoms of a free fatty acid is concentration-dependent [Z] and varies with the viscosity of the medium. In dilute solutions, the T1 values are longer which is indicative of faster rates of motion. This effect is also observed with the monogalactosyldiacylglycerol samples in [2H4]methanol. A comparison of the TXvalues for B. uulguris monogalactosyldiacylglycerol at a range of concentrations (68, 100 and 200 mg/ml) shows a trend to longer values with decreasing concentration (Fig. la and b). The rate of change of T1 values generally increases towards the carbons of the methyl end of the acyl chain, but the overall trend is also apparent in the glyceryl and galactosyl carbon atoms where the relative change in T1 is small and similar to those of the first few carbon atoms of the acyl chain (Fig. lb). These dilution effects must be borne in mind when comparing the relaxation data from the different sources. Specifically, the data for Synechococcus sp. monog~ac~syldia~yl~ycerol yvere obtained using only 80 mg/ml of material and the Ti values seem anomalously long. Comparative studies. The 13C longitudinal relaxation times for the carbon atoms in the monogalactosyldiacylglycerol samples from the four organisms and the hydrogenated B. uulguris monogalactosyldiacylglycerol have been determined and the data are deposited in the BBA Data Bank. In all the monog~a~tosyldiacylglycerol molecules studied, the T1 values of the glyceryl and galactosyl carbon atoms of the headgroup remain constant (the higher values of 0.35-0.55 s for Synechococcus sp. monogaIactosyldiacylglycero1 are due to its lower concentration) with the glyceryl carbon atoms being consistently shorter. Typical values are 0.35 s for the galactosyl carbons and 0.25 s for the glyceryl carbons. The glyceryl chain is held quite rigidly and the galactosyl ring
658
A
12.13x_ 11,14x,
\
-.
. ‘\.
--x_
--__
-_
‘\
--__ ---
IOX- _ ‘x__ 9”-_-‘-.x__;-----‘r--z:==--_
-
----__ _ _--_--_=
-x12,13 ~ll.14 x9.10
MGG
g I=
05-
1
o-4-
Ti 0 a
0.3
-d c -a f t F0.l 4
o-z
Concentration
\
-
---__ 1’. \
-
(mg/ml)
\
---___
I---___ \ *______----=7--c_
._
_----*,
--
---_*3’
-
1 0
I
1
I
I
I
50
68
100
150
200
MGG
Conccntratlon
(mg/ml)
Fig. 1. Dilution effects on longitudinal relaxation times of B. vulgaris monogalactosyldiacylglycerol (MGG). (a) T1 values (in seconds) for the carbons atoms of the acyl chain vs. concentration (mg/ml). (b) T1 values (in seconds) for the headgroup glyceryl and galactosyl carbon atoms vs. concentration (mg/ml).
has only slow, restricted movement regardless of the degree of unsaturation and motions of the esterifying acyl chains. In contrast, the T1 values of the acyl chain carbons vary considerably in the five samples studied. The shortest T1 values, for the equivalent acyl chain carbon atoms are observed for the fully saturated sample with a range of values between 0.45 and 4.2 s. The introduction of a single double bond into one of the acyl chains, as in A. niduluns and Synechococcus sp. monogalactosyldiacylglycerols, increases the T1 values of the carbon atoms of the saturated chain to between 0.37 and 5.4 s. This interaction between saturated and unsaturated acyl chains has been previously noted by us in the plant lipid, sulphoquinovosyldiacylglycerol [4]. The major
659
difference in rates of motion, however, occurs in the unsaturated acyl chain at carbon atoms beyond the double bond where typically, at C-11, the relaxation times may increase from 0.9 to 2.0 s. There is further increase in the T1 values with the introduction of additional double bonds into the chain. Thus the T1 values for the acyl chains of U. Zactuca are consistently longer than those in A. nidulans, indicating that, in U. lactuca, considerably faster motion is occurring. For example, the terminal methyl group T1 value increases from 5.4 to 8.1 s. As is the case in the free fatty acids in chloroform [ 21, and the purified chloroplast lipids of B. uulguris [3--51, this increase in T1 value is related to the segmental motion along the chain. For saturated chains this requires @coupled truns-gauche rotations about C-C bonds while in unsaturated chains the more energetically likely single trunsgauche rotation about the bond adjacent to the double bond is able to induce motion. In effect, the introduction of a double bond into the acyl chain permits more freedom of movement. Polyunsaturated acyl chains should then have even more mobility and this is reflected in the T1 values which show that the rate of motion within the acyl chains of the monogalactosyldiacylglycerol samples increases markedly beyond each double bond. The effect is cumulative and as the degree of unsaturation increases so does the overall fluidity of the acyl chain. At carbon atoms before the position of the double bond, those up to C-S, the T1 values for all the acylating moieties, except for those of the 16 : 4 and the 18 : 4 acids, are very similar and occur in the range of 0.5-1.0 s, again indicating that changes in mobility are primarily induced by the double bonds present. These results on the relative mobilities of the acyl chains of the various monogalactosyldiacylglycerol samples may be compared to monolayer experiments which give information on the packing characteristics of the molecules [ 81. The monolayer behaviour of the different monogalactosyldiacylglycerol samples is not directly related to the inherent motional properties of the monomeric species studied here. The major differences between the fully saturated monogalactosyldiacylglycerol and the unsaturated samples demonstrate that packing in a monolayer is dependent upon the volume of the acyl chains rather than the headgroups and that the introduction of a single double bond is primarily responsible for changes in the packing behaviour. Further unsaturation has very little effect. The similar packing characteristics in the unsaturated samples studied may also reflect the position of the double bond since in all four natural monogalactosyldiacylglycerol samples, the double bond closest to the headgroup occurs between C-8 and C-9. For other monogalactosyldiacylglycerol molecules with their first double bond closer to the headgroup the monolayer pressure vs. area curves may be different from those reported. This dependence on the first double bond for variation of properties is also reflected in the 13C longitudinal relaxation time data where the T1 values only change significantly at carbon atoms at, and beyond, the position of this double bond. The major difference between the monolayer and relaxation time studies is that the packing characteristics depend only on the presence of a double bond, whereas the T1 values change with the degree of unsaturation. The use of the two techniques provides complementary information on the monogalactosyldiacylglycerol molecules, since we obtain knowledge of the static arrangements
660
from the monolayer studies and data on the dynamics of motion and fluidity from 13C-NMR. There is wide variation between the monogalactosyldiacylglycerols of higher plants and that of other species, in the degree of unsaturation and mobility of the acyl chains. Since monogalactosyldiacylglycerol constitutes about half of the polar lipid of the photosynthetic membranes, its behaviour is expected to typify that of the whole membrane bilayer. The range of mobilities found in the acyl chains of monogalactosyldiacylglycerol molecules for different species indicates that the fluidity of the membrane may vary within wide limits without affecting the functional photosynthetic integrity of the system, and indeed, it has been reported that partial hydrogenation of whole chloroplasts has no significant effect on photosynthetic electron transport [12]. The biological rationale for the diversity of unsaturation in monogalactosyldiacylglycerol molecules remains unclear. References Campbell,
I.D.
and
Dobson.
C.M.
(1979)
Methods
Biochem.
Anal.
25,
l-133
Johns.
S.R..
Leslie.
D.R.,
Willing.
R.I.
and
Bishop,
D.G.
(1977)
Aust.
J. Chem.
30,813-822
Johns,
S.R..
Leslie.
D.R.,
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R.I.
and
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30.
823-834
Johns,
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65-72
J.M.,
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Bishop,
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Benson,
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J.H.,
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(1978)
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MacPherson,
AS.
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(Gibbs. Johns,
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129-148,
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