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Electrostatic Layering of Giant Vesicles.

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The XPlor program package [17] was employed to calculate the structure of proteinbound 1 It cooled from 1200 to 50 K within 1 ps with both the geometric and a
relaxation matrix (correlation time for the complex 20 ns) [21] energy terms turned
on. The structures with the smallest R factor were selected.
Received: April 23, 1997
Revised version: July 14, 1997 [Z10376IE]
German version: Angew Chem. 1997, 109,2592-2596
Keywords: molecular recognition
nucleotides proreins R N A
NMR spectroscopy
[I] M. Sprinzl. C. Steegborn, F. Hubel, S. Steinberg. Compilarion of tRNA Sequmce.\ and Sequences ff rRNA Genes, Nucleic Acids Res. 1996, 24, 68-72.
[2] M Taiji, S. Yokoyama, S. Higuchi. T. Miyazawa, J Biochem. 1981, 90, 885888.
131 J. Rudinger. B. Blechschmidt, S Ribeiro, M. Sprinzl, Biochem. 1994,33,56825688.
14) a) J. Jonik, I . Rychlik, J. Smrt, A. HolL, FEES Left. 1979, 98, 329-332; b) J.
Jonik, J. Smrt. A Holy, I. Rychlik, Eur. J Biochem. 1980, 105, 315-320.
[5] L.Servillo, C . Balestrieri, L. Qudgholo, L. lorio, A. Giovane, Eur. J Biochem.
1993.213. 583 589.
[6] B. Nawrot, W. Milius. A. Ejchart, S. Limmer, M. Sprinzl, Nuclric Acids Res.
1997, 25, 94X -~954.
[7] P Nissen. M. Kjeldgaard, S. Thirup, G. Polekhina, L. Reshetnikova. B. F. C.
Clark, J. Nybory. Science 1995, 270, 1464-1472.
(81 0.
Jardetzky. G C. K. Roberts, N M R in Molecular Biology, Academic Press.
Orlando, FL, USA, 1981, pp. 115-142.
[9] L:Y. Lian, G. C. Roberts i n N M R ofMocromolecules(Ed.:G. C. K. Roberts),
IRL, Oxford, 1993, pp. 153-182.
[lo] M. Taiji, S. Yokoyama, T. Miyazawa, J Biochem. 1985, 98, 1447-1453.
I1 I ] M. Sprinzl. M. Kucharzewski, J. B. Hobbs. F. Cramer, Eur. J. Biorhem. 1977,
78. 5 5 ~61
[12] F. Janiak. V. A. Dell, J. K . Abraharnson, B. S. Watson, D. L. Miller, A. E.
Johnson, Blochem. 1990,29,4268-4277.
[I31 A. P. Campbell. B. D. Sykes, Annu. Reis. Biophys. Biomol. Strucr. 1990, 29,
99 122
I141 a ) F. NI. H A. Scheraga, Ace Chem Res. 1994, 27, 257-264; b) F. Ni. Prog.
N M R, 26, 517-606.
[15] D. Neuhaus. M. Williamson, The Nuclear Overhauser Effect in Strucrural and
Confor.mutiodAnalysis. VCH, New York, 1989.
[16] a ) H. Berchtold, L. Reshetnikova, C 0. A. Reiser, N. K Schirmer, M. Sprinzl.
R. Hilgenfeld. N a m e 1993, 36S, 126-132; b) M Kjeldgaard, P. Nissen, S.
Thirup. J. Nyborg, Sfructure 1993, 1 . 35-50.
[17] A. T. Brunger. X-Plor, Version 3.843, 1996
1181 S. Limmer, C. 0 A. Reiser, N. K. Schirmer, N. W. Grillenbeck. M. Sprinzl,
Biodii~m 1992. 32, 2970-2977.
[I91 A. V. Azhayev. S. V. Popovkina, N. B. Tdrussova, M. P. Kirpichnikov. V. L.
Florentiev, A. A. Krayevsky, M. K. Kukhanova, B. P. Gottikh, Nucleic Acids
Rc~.s.1979. 7. 2223 ~2234.
[20] H:P. Vornlocher. W.-R Scheible. H. G. Faulhammer, M. Sprinzl, Eur. J.
Bioc.hem. 1997. 243. 66- 71.
[21] C . R. Cantor. P. R. Schimmel, Biophysical Chemistr?. Part II, W. H. Freeman,
San Francisco. 1980, p. 461
1221 P J Krauhs. .I. A p p l Crysrallogr. 1991. 24, 946-950.
Electrostatic Layering of Giant Vesicles **
controlled. We report here experiments in which a giant vesicle,
an object of considerable recent attention,[’ 61 is successfully
coated with lipid bilayers. The layering is electrostatically driven
and detectable under phase-contrast microscopy. Controlled
layering now joins growth, fusion, undulation. excretion, and
healing15. as observable phenomena with giant vesicles.
Giant anionic vesicles (20-50 pm in diameter) were prepared
from a mixture of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the sodium salt of 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-rac-(l-gIycerol)](POPG), and cholesterol
in a molar ratio of 79:9: 12. Giant cationic vesicles were prepared from POPC, didodecyldimethylammonium bromide
(DDAB), and cholesterol (molar ratio also 79 :9 : 12). Vesicle
formation was carried out with the following protocol: Lipid
mixtures in CHCIJMeOH were stripped of solvent, and the
resulting films vortexed with deionized water. Lyophilization
gave fluffy white powders. Afterwards, the powder (0.1 mg or
less) was smeared onto the microscope slide (within the confines
of a Teflon O-ring cemented onto the slide). The sample was
hydrated with water ( ~ 0 . mL)
for at least 30 min to give a
polydisperse array of vesicular structures.
Giant vesicles of opposite charge were brought into contact
according to the following procedure: A suitable anionic vesicle
was grasped with a polished holding pipet (under slight suction
provided by a picoinjector device) and guided into a vesicle-free
region of the microscope field. Cationic vesicles from another
preparation were then withdrawn into a capillary pipet with the
aid of a hand-controlled microsyringe. This pipet was emptied
of its contents adjacent to the lone anionic vesicle. Finally, the
held anionic vesicle was brought into contact with a “free”
cationic vesicle, and the interaction between the two membranes
was observed as a function of time under 400-fold magnification. Others have shown that light defracted from even a single
bilayer is visible by light micros copy,^'] and that vesicle adhesion
is amenable to micromechanical testing.[’]
Neutral vesicles composed only of POPC and cholesterol
failed to “stick” even when repeatedly pushed into contact; the
free vesicle immediately diffused away from the one held stationary by the pipet. In contrast, two vesicles of opposite charge
“snapped” together (Figure 1). The cationic vesicle formed a
Figure 1. A) A “free” cationic vesicle that adheres to an anionic vesicle held by a
micropipet. B) After about 9 0 s the cationic vesicle coats itself with anionic lipid
from the stationary vesicle and diffuses away. Bar = 25 pm
Fredric M. Menger* and Jason S. Keiper
In the Langmuir-Blodgett method, a vertical plate is dipped
up and down through a water-supported monolayer, thereby
depositing monolayers onto the plate one a t a time.“] Thus, the
thickness and constitution of a multilayered assembly can be
[*I Prof. F. M . Mcnger, J. S. Keiper
Department of Chemistry
Emory University
Atlanta. GA 30312 (USA)
Fax Int. code +(404)727-6586
[**I This work wiib supported by the U. S. National Institutes of Health
Angm Chm7 Inr Ed En@ 1997, 36. No 22
concavity on its surface in order to enhance its contact with the
neighboring anionic vesicle. The configuration was, however,
unstable. After approximately 90 s the cationic vesicle departed
while concurrently “peeling” lipid away from the anionic vesicle, as evidenced by a decided thinning of the anionic membrane. When the same two vesicles were forced into contact
following their separation, they were unable to adhere. Appai-ently, the transfer of lipid from anionic to cationic vesicle generated two multilamellar vesicles, both with outer anionic shells
that repelled each other.
WILEY-VCH Verldg GmbH, D-69451 Weinhelm, 1997
0570-0833/97/3622-2489 S 17 SO+ 50 0
To confirm the above conclusions, the held vesicle from the
preceding experiment was brought into contact with a new
cationic vesicle. Adhesion occurred as with the first cationic
vesicle (Figure 1). This is strong additional evidence that the
anionic vesicle never lost its negative charge, and that lipid
transfer was, therefore, unidirectional. The cationic vesicle must
have thereby become coated with one or more of the anionic
bilayers (the exact number is being now investigated by a newly
designed Oxford Institute scanning transmission electron
micropscope cryoprobe) . If, instead, this receptor vesicle had
actually incorporated anionic lipid within its outer cationic
bilayer, its surface area would have greatly expanded; this was
not observed. Moreover, such a direct transfer of lipid between
bilayers should be slow as is “flip-flopping” between Ieaflets of
a single bilayer.Igl
A cationic vesicle will frequently burst upon touching an anionic vesicle. Figures 2A and 2B show a small cationic vesicle
that adhered to a larger anionic vesicle and then, upon bursting,
coated only the “northern” hemisphere of the host vesicle (see
the enhanced thickness of the coated region in Figure 2B). Subsequently, a second cationic vesicle fastened to an uncoated
section of the anionic vesicle (Figure 2C). This cationic vesicle
also burst to coat the remaining portion of the anionic vesicle
(Figure 2D). When this anionic vesicle, now encapsulated with
at least one cationic bilayer, was brought into contact with a n
anionic vesicle of nearly equal size, the latter adhered and burst
(Figure 2E). The resulting lipid wrapped around the host membrane (Figure 2F), further thickening it. The twice-altered vesicle must have regained its negative charge because it could now
no longer adhere to anionic vesicles.
The reproducible negative-positive-negative sequence in Figure 2 is reminiscent of the lamination possible with Langmuir-
Blodgett films. The vesicle system has a n operational advantage
over films, however, in that films are blocked on one side by a
solid support; this makes studies of diffusion through films
more difficult. In any event, periodicity in vesicle membranes (as
in solid films)[“] could lead to cooperative electronic effects
and, thus, to systems with useful electrical, magnetic, and optical properties.
Servuss and Boroske”’] showed years ago that a membrane
image can be scanned for intensity as a function of position as
one passes through the membrane. We now applied this method
to Figure 2 with the aid of a n Image-Pro Plus processing routine. Figure 3A gives such an intensity distribution as the held
vesicle (just prior to the adhesion shown in Figure 2A) is
scanned along the indicated arrow. Intensities of43 and 47 units
correspond to the two points at which the arrow crosses a presumed single bilayer. After the small vesicle bursts and coats the
northern hemisphere of the large vesicle (see Figure 2B), the
scan changes to that in Figure 3B. The intensities are now 58
and 95 units, which correspond to single and double bilayers,
respectively. The scan of Figure 2 F (not shown) indicates that
both crossover points are now triple bilayers (118 units).
Figure 3. A) Intensity profile of the vesicle membrane prior to the adhesion shown
in Figure 2A. The two minima represent the points where the white arrow in the
micrograph crosses the bilayer. B) Intensity profile of the partially coated vesicle
shown in Figure 2B.The intensity differences are measured from minima to adjacent maxima (representing the white “halo”) and are in arbitrary units.
Electrostatic charge is a n important feature of cell membranes,[’2’ and vesicles provide a useful means for evaluating
this property. Most corresponding model studies in the past
were carried out on submicroscopic vesicles (only 20-200 nm in
diameter), for which information must be inferred spectroscopi~ally.[’~*
I4I Giant vesicles, on the other hand, allow the surface
charge to be controlled while the modifications are examined
directly by optical methods.
Received: April 2,1997 [Z10303IE]
German version. Angew. Chem. 1997, 109, 2602-2604
Keywords: cytomimetic chemistry
microscopy * vesicles
Figure 2. A) Adhesion of a small cationic vesicle to a large anionic vesicle.
8) Within 10 s the cationic vesicle bursts and coats the northern region of the
anionic veslcle (indicted by the arrows). C ) ,D) A second cationic vesicle binds,
bursts after 2s, and coats the entire anionic vesicle. E),F) An anionic vesicle
binds to the new cationic surface, bursts, and regenerates an anionic surface.
Bar = 25 pm.
a WILEY-VCH Verlag GmbH, D-69451 Weinheirn, 1997
electrostatic interactions
[l] D. G. Zhu, M. C. Petty, H. Ancelin, J Yarwood, Langmuir 1992, 8, 619.
[2] U. Seifert, Adv. f h p . 1997, 46, 13.
131 K. Berndl, J. Kas, R. Lipowsky, E. Sackmann, U. Seifert, Europhys. Lett. 1990,
13, 659.
[4] H.-G. Dobereiner, E. Evans, M. Kraus, U . Seifert, M. Wortis, Phyx Rev. E
1997, 55,4458.
0570-0833/97/3622-2490 $17.50+.50/0
Angew. Chem. Int. Ed. Engl. 1997,36, No. 22
[S] F. M Menger. K. D. Gabrielson,
Angew. Chem.
1995, 107. 2260, Angeu. Chem. Inr. Ed. EngL
1995. 34, 2091
[6] F. M. Menger. S J. Lee, Langmuir 1995, f I, 3685.
[7] H. Ringsdorf. B. Scbarb, J. Venzmer, Angew.
Chem. 1988.100,117; Angew. Chem. Inr. Ed. Engl.
1988. 27. 113.
[8] E. Evans, D. J Klingenberg, W. Rawicz, F. Szoka,
Lungmuir 1996. f2. 3031.
[9]R. D. Kornberg, H. M. McConnell. Biochemistry
1971. IO. 1111
[lo] T E. Mallouk. H. Lee, 1 Chem. Educ. 1990, 67,
I l l ] R.-M. Servuss. E Boroske. Chem. Phys. Lipids
1980, 27. 57.
[12] G. Deng. T. Dewa. S . L. Regen,.T Am. Chem. Soc.
1996, f i x . 8975
[13] S. Ohki. S. Roy. H. Ohshima. K. Leonards, Biochrmistrr 1984, 23, 6126.
[14] L. Stamatatos, R. Leventis, M. J. Zuckermann,
J R. Silvius. Biochemutry 1988, 27, 3917.
2) ZnBr2
B (X = Br)
E (branched, R f H)
Addition of Azaenolates to Simple,
Unactivated Olefins”“
Katsumi Kubota and Eiichi Nakamura*
G (linear, R f
Scheme 1. Addition of a zinc hydrazone to a simple olefin; E = H, H,C=CHCH,,
Whereas an enolate anion and its azaenolate congener readily
add to a carbonyl compound,[’] they do not normally react with
a simple, unactivated olefin;[21little effort has been expended to
effect such an “olefinic analogue” of the aldol r e a c t i ~ n . [We
entry 1). This indicates that the y-zinc hydrazone D (a y-enolate
now found that addition of zinc hydrazone C to a simple olefin
or “bis(homoenolate)”)[7a]does not isomerize to a more stable
takes place at room temperature to afford the organozinc compound D, which then reacts with an added electrophile to give E
isomer, such as F. We did not observe the formation of products
in good yields (Scheme 1). The overall process represents a
due to addition of D to the C=N bond of D or E, or to
three-component synthesis of carbonyl derivatives. The key to a
further reaction of D with ethylene. There was no sign of butylsuccessful reaction is the use of the BuZn” species C instead of
group transfer from C in any of the reactions examined.[81The
the BrZn” species B, with which the reaction was impractically
use of the butylated species C is crucial, since B afforded E
(E = H) in much lower yield (22%) under the same reaction
N,N-Dimethylhydrazones A are ubiquitous synthetic substitutes of ketones, as they can be readily synthesized from and
Cyclic (entries 2 and 6) and acyclic ketone hydrazones (enconverted back into the corresponding ketonesc5’The addition
tries 1, 3-5, 7- 13) reacted equally well. Deprotonation of an
reaction of the zinc hydrazone to an isolated olefin was carried
unsymmetrical ketone hydrazone (entry 5 ) took place at the less
out in the following manner for the N,Ndimethylhydrazone of 1,5-diphenylpentan-3R
one (see Experimental Section for details): The
hydrazone was treated in diethyl ether sequen1: R = H
4: R = 2-CF&H4
tially with one equivalent each of tBuLi, ZnBr,
5: R = CH3(CH&
(to generate B), and BuLi (to generate C).E6’
3: R = 4-CH30C6H4
6: R = (C4H9)3SnCH2
The solution of C was then placed under an
ethylene atmosphere (5- 10 atrn) and stirred
for four days at 20-35 “C. After the pressure
N- NMe2
had been released, water or D,O was added to
afford the ethylated product E in 88% yield
(E = H or D. R = H ; 100% deuterium incorporation at the terminal carbon atom; Table 1,
[*I Prof. E. Nakamura, Dr. K. Kubota
Department of Chemistry
The University of Tokyo
Bunkyo-ku. Tokyo 113 (Japan)
F a x . Int. code +(3)5800-6889
e-mail nakamura(
“*I We thank the Japanese Ministry of Education, Science, Sports. and Culture for financial support on the
research. K. K. thanks the Japan Society for the Promotion of Science (JSPS) for a predoctoral fellowship.
Angew Chem Inr. Ed. Engl. 1997. 36, No. 22
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