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Langmuir 1998, 14, 2796-2800
Protein-DNA Double and Triple Layers: Interaction of Biotinylated DNA Fragments with Solid Supported Streptavidin Layers Kuniharu Ijiro*,† and Helmut Ringsdorf Institut fu¨ r Organische Chemie, Universita¨ t Mainz, J.-J.-Becherweg 18-20, D-55099 Mainz, Germany
Eckhard Birch-Hirschfeld, Siegfried Hoffmann, Ute Schilken, and Michael Strube Institut fu¨ r Biochemie, Universita¨ t Halle, Weinbergweg 16a, D-06120 Halle/Saale, Germany Received December 9, 1997. In Final Form: February 27, 1998 The specific interaction of streptavidin with biotinylated lipids at the air-water interface leads to a formation of optically anisotropic two-dimensional streptavidin (2-D) crystals, where two of the original four biotin-binding sites remain free. These assembled streptavidin matrixes were used as a template for docking of double-stranded oligonucleotides biotinylated at a terminal or a centered position. A biotinylated lipid monolayer was deposited on an electrode of a quartz crystal microbalance (QCM), and docking processes of the protein and the oligonucleotides were detected as frequency changes related by mass changes on the QCM. The bis-biotinylated double-stranded oligonucleotides bound to the primary streptavidin layers made it possible to engineer protein-DNA-protein triple layers. Hydrolysis by a restriction endonuclease indicates that the biotinylated DNA bound to the streptavidin layers remains bioactive.
* To whom correspondence should be addressed: Telephone: +81-11-706-2895. Fax: +81-11-706-4974. E-mail: kijiro@ imdes.hokudai.ac.jp. † Present address: Research Institute for Electronic Science, Hokkaido University, 060 Sapporo, Japan.
the biotinylated lipid monolayer can thus act as a template to immobilize other functionalized biotin molecules,14-17 or bis-biotin couplers can be used to bridge two SA layers.18 Herein we propose a controlled formation of assembled protein-DNA layers based on oligonucleotides. Oligonucleotides offer several advantages over the previous linker molecules. For example, programmed base sequences with an appropriate binding functionality may be prepared automatically with a DNA synthesizer. The interlayer distances of the supramolecular structures can be controlled by not only the length of oligonucleotides but also by the position of a biotin moiety. A utility of DNA for a preparation of new biomaterials and hybrid materials has already been recognized. Previous investigations were focused on designing nanometersized structures including double helices, triplexes, knots, and polyhedra with well-defined geometric shapes of oligonucleotides.19-22 Recently assemblies of Au nanoparticles by hybridization of thiol-terminated oligonucleotides has been described.23,24
(1) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (2) Chaiet, M.; Wolf, F. J. Arch. Biochem. Biophys. 1964, 106, 1. (3) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1. (4) Weber, P. C.; Ohlendorf, D. H.; Wendolowski, J. J.; Salemme, F. R. Science 1989, 243, 85. (5) Green, N. M. In Avidin-Biotin Technology; Bayer, E. A., Wilchek, M., Eds.; Academic Press: San Diego, CA, 1990 (Methods Enzymol. 1990, 184, 51). (6) Vaknin, D.; Als-Nielsen, J.; Piepenstock, M.; Lo¨sche, M. Biophys. J. 1991, 60, 1545. (7) Lo¨sche, M. In Synthetic Microstructures in Biological Research; Schnur, J. M., Peckarar, M., Eds.; Plenum Press: New York, 1992; p 91. (8) Lo¨sche, M.; Peckarar, M.; Vaknin, D.; Als-Nielsen, J. Thin Solid Films 1992, 210/211, 659. (9) Vaknin, D.; Kjaer, K.; Ringsdorf, H.; Blankenburg, R.; Piepenstock, M. Diederich, A.; Lo¨sche, M. Langmuir 1993, 9, 1171. (10) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214. (11) Ahlers, M.; Mu¨ller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 1269.
(12) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, D. Biophys. J. 1991, 59, 387. (13) Hendrickson, W. A.; Pa¨hler, A.; Smith, J. L.; Satow, Y.; Merritt, E. A.; Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2190. (14) Ebato, H.; Herron, J. N.; Mu¨ller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. Angew. Chem., Int. Ed. Engl. 1992, 31, 1087. (15) Herron, J. N.; Mu¨ller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992, 8, 1413. (16) Morgan, H.; Taylor, D. M. Biosensors 1992, 7, 405. (17) Ebersole, R. C.; Miller, J. A.; Moran, J. R.; Ward, M. D. J. Am. Chem. Soc. 1990, 112, 3239. (18) Fujita, K.; Kimura, S.; Imanishi, Y.; Rump, E.; van Esch, J.; Ringsdorf, H. J. Am. Chem. Soc. 1994, 116, 5479. (19) Seeman, N. C. Mater. Res. Soc. Symp. Proc. 1993, 292, 123. (20) Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1994, 22, 5530. (21) Niemeyer, C. M. Angew. Chem., Int. Ed. Engl. 1997, 36, 585. (22) Kool, E. T. Chem. Rev. 1997, 97, 1473. (23) Mirkin, C. A.; Letsinger. R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607.
Introduction The molecular architecture and the molecular engineering of ordered supramolecular systems conjugating biomolecules such as proteins, lipids, sugars, and nucleic acids is interesting from different viewpoints, e.g., supramolecular science, membrane biology and biotechnology. Self-assembled monolayers of proteins formed at lipid monolayers and liposome surfaces have been studied using biotin-avidin or suger-lectin interaction.1 The specific interaction2-5 of streptavidin (SA) with biotin-attached lipids spread at the air-water interface6-9 leads to a formation of optically anisotropic two-dimensional (2-D) streptavidin crystals, where two of the original four biotinbinding sites remain free.10-13 The SA layer docked on
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Figure 2. Hybridization and dehybridization of (a) Exo-36mer and (b) Endo-36mer.
Endo, below the melting temperature (Figure 2). The double-helices are so rigid that geometric distances between two biotin units of ds-Exo DNA depend on the number of sequenced base-pairs. In contrast to the dsExo DNA, the ds-Endo oligonucleotides have an identical distance between two biotin units even though the number of base-pairs is different. Experimental Section
Figure 1. Schematic illustration of (a) docking of streptavidin to a biotin-lipid monolayer deposited on a QCM plate, (b) binding of a biotinylated double-stranded oligonucleotides to the first SA layer, (c) secondary docking of streptavidin to the DNA layer, and (d) hydrolysis of the oligonucleotides by nuclease.
Our experiments to form protein-DNA multilayers using streptavidin monolayers and bis-biotinylated doublestranded oligonucleotides are schematically outlined in Figure 1. The biotin lipid monolayer formed at the airwater interface was deposited on an electrode of a quartz crystal microbalance (QCM), and each docking step was detected as frequency changes of the QCM. QCMs are known to provide very sensitive mass measuring devices because their resonance frequency decreases upon an increase of a given mass on the QCM on a nanogram level.25-31 The multilayer assemblies were prepared by stepwise docking streptavidin and bis-biotinylated DNA onto the solid-supported biotin lipid monolayer as an initial layer. It is important to note that bis-biotinylated DNA layers prepared on the SA layers can be decomposed by an enzymatic hydrolysis with an endonuclease. Chemical formulas of biotin-terminated oligonucleotides (Exo12mer and -36mer) and biotin-centered oligonucleotides (Endo12mer and -36mer) are shown in Scheme 1. The spacers between the biotin units and oligonucleotides were long enough to allow the binding of streptavidin. Since base-sequences of these oligonucleotides are self-complementary, single-stranded oligonucleotides can hybridize themselves to form double-stranded DNA, ds-Exo and ds(24) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609. (25) Okahata, Y.; En-na, G.; Ebato, H. Anal. Chem. 1990, 62, 1431. (26) Okahata, Y.; Ebato, H. Trends Anal. Chem. 1992, 11, 344. (27) Ebara, Y.; Okahata, Y. Langmuir 1993, 9, 574. (28) Tamiya, E.; Suzuki, M.; Karube, I. Anal. Chem. Acta 1989, 217, 321. (29) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58, 1206. (30) Guilbault, G. G. Anal. Chem. 1983, 55, 1682. (31) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355.
Apparatus. Lipid monolayers were prepared by using a LB trough (KSV, Norway, Model 5000). The QCM employed was a crystal of commercially available 9-MHz, AT-cut quartz plate on which Au electrodes were deposited on both sides (area: 15.9 mm2). The quartz plate was assembled in a Teflon holder by vacuum pressure to prevent short circuits in buffer solutions. Electrodes on the QCM plate were connected to a handmade oscillator designed to drive the quartz at its resonance frequency in an aqueous solution. The frequency changes were followed each 10 s by a universal frequency counter (Iwatsu Co., Japan, Model SC 7201) attached to a microcomputer system (IBM). A calibration by casting of polystyrene solutions showed that a frequency decrease of 1 Hz corresponded to a mass increase of 1.05 ng on the QCM electrode (15.9 mm2) both in an aqueous solution and in the air,32 according to a Sauerbrey’s equation.33 The surface of the QCM electrodes was hydrophobized by casting of a silicone thin film from the 2-propanol solution (SERVA). Following measurements were carried out in tris-buffer solutions (Tris/HCl, 10 mM, pH 7.0). Hydrolysis of the ds-DNA by restriction endonuclease Hha I (Sigma) was detected in the trisbuffer solution containing 10 mM MgCl2, 50 mM NaCl, and 1 mM dithiothreitol. Chemicals and Materials. Biotinylated lipid (1) was synthesized as described elsewhere.14 Streptavidin (SA) was kindly supplied by Dianova (Germany) and used without further purification. Four types of biotinylated oligonucleotides (Exo12mer, Exo-36mer, Endo-12mer, and Endo-36mer) were synthesized on the DNA synthesizer (Applied Biosystems, Model 380B) with commercially available reagents (Scheme 1).34 Basesequences of these oligonucleotides are self-complementary.35 Measured melting temperatures (Tm) of Exo-12mer, Exo-36mer, Endo-12mer, and Endo-36mer in the experimental buffer solutions by conventional UV absorption method were 56, 65, 49, and 56 °C respectively, so that they hybridized from single strands (ss-) to form double strands (ds-) in the buffer solutions at the experimental temperature as shown in Figure 2. The biotinylation of oligonucleotides might enhance stability of double helical structures due to hydrophobic moieties of biotin and the spacers. Thus each ds-Exo-12mer and -36mer has two biotin units modified at both terminal of the ds-DNA, and each ds-Endo-12mer and -36mer has two biotin units at the middle positions of the dsDNA (Figure 2). Chloroform (99.8%, spectrophotomeric grade, Aldrich), NaCl (99.5%, BioChemika, Fluka), MgCl2 (98%, anhydrous, Fluka), and dipalmitoyl L-R-phosphatidylethanolamine (DPPE) (98%, Sigma) (2) were used as received without purification. The tris(32) Okahata, Y.; Shimizu, O. Langmuir 1987, 3, 1171. (33) Sauerbrey, G. Z. Phys. 1959, 155, 206. (34) Keller, G. H.; Manak, M. M. DNA Probes; M Stockton Press: New York, 1989; p 105. (35) Yoon, C.; Prive, G. G.; Goodsell, D. S.; Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6332.
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Ijiro et al. Scheme 1
buffer solution was prepared by TRIZMA PRE-SET CRYSTALS (Sigma). Deionization water from a Milli-Q (18 MΩ-cm) system was used to prepare all aqueous solutions.
Results and Discussion Deposition of Biotin-Lipid Monolayer. A mixture of biotin-lipid (1) and DPPE (2) as a matrix component (1:2)5:95 in molar ratio) were spread from chloroform solution on pure water at 20 °C.14 The formed monolayer was compressed at a speed of 0.06 nm2molecules-1 min-1. The QCM plate with the holder was perpendicularly pulled down into the subphase at a speed of 50 mm min-1 to deposit the formed monolayer on the QCM electrode at a surface pressure of 35 mN m-1. A frequency change of -45 ( 2 Hz in water, which corresponded to the mass increase of 47 ( 2 ng due to a deposition of the mixed biotin lipid, was in good agreement with the calculated mass of the condensed lipid monolayer by using a mean molecular area (0.45 nm2 molecules-1) at the air-water interface and a mean molecular weight (700 g mol-1) of the mixed lipids (Table 1). Then the QCM holder was assembled in a measurement cuvette (3 mL) in the subphase to prevent the surface of deposited monolayer exposing to the air. The QCM holder and the cuvette
were put in a thermally controlled chamber at 22.1 °C and QCM measurements were carried out with a stirring microchip. Docking of First Streptavidin (SA) onto the Biotin Lipid Monolayer. As a defined amount of streptavidin solution (50 mM) was added into the cuvette with a microsyringe, the frequency changes due to the binding of streptavidin onto the biotin lipid monolayer were followed as a function of time as shown in Figure 3 a. The frequency change was saturated at -52 ( 3 Hz when the streptavidin concentration was increased to 800 nM as shown in Figure 4 a. This frequency change was converted to mass increase (∆m) of 55 ( 3 ng (0.92 ( 0.05 pmol) on the electrode of 15.9 mm2. The electron crystallographic experiments showed that a mean area of crystallizing streptavidin on the biotin lipid was 2500 Å2 per one molecule.12 Thus the resulted mass increase of binding streptavidin almost agrees with the estimated mass of streptavidin monolayer (63 ng) from the crystallographic data. Streptavidin could not bind to a monolayer without the biotin lipid at the same concentration. This indicates that a condensed streptavidin monolayer was formed on the biotin-lipid monolayer by the specific biotin-streptavidin interaction. When the binding of streptavidin on
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Table 1. Step-by-Step Formation of Lipid-SA-DNA-SA Layers on the QCM docking molecule
docked layer
-∆F (Hz)
∆m (ng)
∆m (pmol) 68 ( 4 (3.4 ( 0.2)a
lipid monolayer
QCM plate
45 ( 2
47 ( 2
SAb
lipid monolayer
52 ( 3
55 ( 3
0.92 ( 0.05
10 ( 1 11 ( 1 22 ( 1 22 ( 1
11 ( 1 12 ( 1 23 ( 1 23 ( 1
1.4 ( 0.1 1.5 ( 0.2 1.0 ( 0.1 1.0 ( 0.1
50 ( 2 47 ( 18 55 ( 5 58 ( 13
53 ( 2 49 ( 19 58 ( 5 61 ( 14
0.88 ( 0.04 0.82 ( 0.31 0.96 ( 0.09 1.0 ( 0.22
Exo-12merc Endo-12merc Exo-36merc Endo-36merc
SAe
SA Exo-12mer Endo-12mer Exo-36mer Endo-36mer
Ka (M-1)
molar ratio
4.9 × 107
3.2 × 108 3.6 × 105
1.5:1d 1.6:1d 1.1:1d 1.1:1d 1.0:1f 0.9:1f 1.1:1f 1.1:1f
a ∆m corresponds to the 5% biotin lipid 1 in the layer. b At a concentration of 800 nM. c At concentrations between 110 and 130 nM. Molar ratio of the docking molecules against streptavidin in the docked layers. e At concentrations between 10 and 12 µM. f Molar ratio of streptavidin as docking molecules against streptavidin in primary protein layers.
d
Figure 3. Frequency changes of a biotin lipid monolayer on gold (according to Figure 1) after injections of (a) a streptavidin solution (500 nM), (b) a ds-Exo-36mer solution (110 nM), and (c) a steptavidin solution (4.2 µM).
Figure 4. Titration curves according to Figure 1 of (a) first streptavidin, (b) ds-Exo-36mer, and (c) second streptavidin.
the biotin-lipid monolayer was saturated, the molar ratio of streptavidin molecule to the biotin-lipid 1 molecule (5 mol % of the monolayer) was estimated to be 0.27:1. This means that one molecule of streptavidin occupied about three molecules of the biotin-lipid 1 at the docked monolayer. As streptavidin has two binding pockets at one side, this binding process progressed highly efficiently. Further, a binding constant between streptavidin and the biotin lipid at the monolayer was calculated as 4.9 × 107 M-1 by a Lineweaver-Burk plot (1/∆m versus 1/[streptavidin]) of a titration experiment (Figure 3a). Although the binding constant between D-biotin and streptavidin has been reported2,5 as 1015 M-1, the binding constant
between the biotin lipid and streptavidin at the monolayer might be reduced because of an amidation of a carboxyl group of D-biotin and a steric possible hindrance around the biotin moiety. Binding of Biotinylated Oligonucleotides onto the First SA Layer. After the preparation of the SA layer, the solution in the cuvette was exchanged for fresh buffer solution, and a defined amount of biotinylated oligonucleotide solutions (50 µM) was added. The biotinylated oligonucleotides were expected to dock to the free outerbinding pockets of the primary SA layer. Typical frequency changes after injection of the ds-Exo-36mer solution were shown in Figure 3b. An injection of the same amount of nonbiotinylated ds-36mer instead of the biotinylated ds-Exo-36mer did not change the frequency at all. When a D-biotin solution was pre-added to the primary SA layer, the following biotinylated oligonucleotides did not bind to that layer. These indicate that the biotinylated oligonucleotides dock to the SA layers by means of the specific biotin-streptavidin interaction. The binding constant of ds-Exo-36mer to the first SA layer was calculated as 3.2 × 108 M-1 by the Lineweaver-Burk plots of the titration experiments (Figure 4b). This value was 10 times higher than that of streptavidin for the biotin lipid monolayer because of the lower steric hindrance of the DNA compared with that of streptavidin (Table 1). The frequency changes after injections of the ds-Exo-36mer solutions were saturated at the concentration of 200 nM and mass increase reached 30 ( 1 ng (1.4 ( 0.1 pmol) as shown in Figure 4b. At the saturated binding, the molar ratio of ds-Exo-36mer to streptavidin was 1.5:1. Although one molecule of streptavidin bound to the biotin lipid monolayer has two free outer-binding pockets, ionic repulsion among DNA moieties themselves might disturb the formation of condensed oligonucleotides layers. The binding time courses of ds-Exo-36mer (110 nM) and dsEndo-36mer (100 nM) to the primary SA layers were shown in Figure 5 as frequency changes. The binding speed of the ds-Endo-36mer was slower than that of the ds-Exo-36mer because of a steric hindrance around biotin moieties. In both cases, however, the frequency changes (∆F) reached the same values as expected for DNA of the same molar mass. Table 1 shows frequency changes (∆F), converted mass increases (∆m) for the various biotinylated oligonucleotides bound to the first SA layers at the concentration between 110 and 130 nM and estimated molar ratios of the binding DNA per one streptavidin molecule. The short oligonucleotides, ds-Exo-12mer and ds-Endo-12mer, bound to the SA layers with higher molar ratios than the long oligonucleotides, ds-Exo-36mer and ds-Endo-36mer, at the
2800 Langmuir, Vol. 14, No. 10, 1998
Figure 5. Time-courses of frequency changes after injections of the ds-Exo-36mer solution (110 nM, closed circuit) and the ds-Endo-36mer solution (100 nM, open circuit) to the first SA layers (see Figure 1b).
same concentration. The stronger ionic repulsion was considered to reduce the binding affinity of the longer biotinylated oligonucleotides. As compared with Exo- and Endo-type DNA, the position of biotin modified on the nucleotides did not influence the binding amount under these concentrations of oligonucleotides. Secondary Binding of Streptavidin to Biotinylated Oligonucleotide Layers. The ds-biotinylated oligonucleotides, which have two biotin moieties in each double strand, can dock with SA layers in two different ways; (i) one biotin unit of the DNA docks at the SA layer leaving one biotin unit free and (ii) both of the biotin units will dock at the primary SA layer. Additional binding behavior of streptavidin to the streptavidin-DNA double layer can reveal the binding manner of the biotinylated ds-DNA to the SA layers. After the preparation of the DNA layers, the solution in the cuvette was exchanged for fresh buffer solution. The frequency changes after the injection of the secondary streptavidin solutions to the Exo-36mer layer was shown in Figure 3c. The frequency changes due to the binding of the second streptavidin reached from -50 to -58 Hz for all biotinylated DNA layers (Table 1). The mass of the second SA layers were almost identical with those of the first SA layers. This indicates that in all cases one of the biotin moieties of the DNA had remained free when the biotinylated ds-DNAs were bound to the first streptavidin layers. The binding speed of the second streptavidin was much slower than that of the first streptavidin as shown in Figure 3a,c. The binding constant of the second streptavidin to the Exo-36mer layer was calculated as 3.5 × 105 M-1 from the titration curve in Figure 4c, and it was 100 times lower than that of the binding constant of streptavidin to the original biotin-lipid monolayer (4.9 × 107 M-1) as shown in Table 1. Although the immobilization of DNA on the first SA layer inhibited the binding affinity of the biotin unit, the final molar ratios of the second streptavidin bound to the DNA layers against the first streptavidin bound to the biotin lipid layers were almost 1:1. It could be concluded that the supramolecular structures of the lipid-protein-DNA-protein were formed by the biotin-streptavidin interaction. DNA was sandwiched between SA layers. Hydrolysis of the Oligonucleotide Bound to the SA Layer. Enzymatic hydrolysis of the oriented DNA bound to the first SA layer was investigated. After
Ijiro et al.
Figure 6. Frequency changes of a biotin lipid monolayer after injections of (a) a streptavidin solution (500 nM) and (b) a dsExo-36mer solution (110 nM) and (d) hydrolysis of the bound oligonucleotide after addition of an endonuclease Hha I solution (a tris-buffer solution containing 10 mM MgCl2, 50 mM NaCl and 1 mM dithiothreitol) (see schematic representation in Figure 1d).
preparation of the ds-Exo-36mer layer on the SA layer, endonuclease Hha I solution was added into the solution. Hha I is a endonuclease which can hydrolyze a doublestranded 5′GCG/C3′ sequence specifically.36 Doublestranded Exo-36mer has two parts of the substrate sequence, and ds-Exo-36mer bound to the first streptavidin layer is expected to be hydrolyzed enzymatically by an endonuclease Hha I (Figure 1d). Figure 6d shows that the mass on the QCM decreased immediately after an injection of Hha I. The decreased mass was corresponded to 64% of ds-Exo-36mer bound to the first SA layer. It was consistent with hydrolysis and release at a close sequence of 5′GCGCGC3′ to the first SA layer. This indicates that the oriented oligonucleotides remain their bioactivity even when bound to streptavidin. This could be further shown by the specific binding of the FIS protein, one of the DNA recognizing proteins, to the oriented oligonucleotides docked on the SA layer. Conclusions The supramolecular multilayer system of the lipidprotein-DNA-protein was formed by the strong and specific biotin-streptavidin interaction. Each binding process was detected by QCM as the frequency changes related to mass changes in a nanogram level. The position of the biotin-unit modified on the double-stranded oligonucleotides might give rise to a different oriented binding of the oligonucleotides to the SA layerssnot further investigated in this paper. Such oriented-DNA fragments may be useful to investigate the mechanisms of biointeraction with DNA on a molecular level, e.g., specific binding of FIS-protein to the oriented DNA docked on the SA layers. This has already been investigated and will be published elsewhere.37 Acknowledgment. We are very grateful to the Alexander von Humboldt Foundation, 53173 Bonn, Germany, for their support of this research. LA971352V (36) Roberts, R.; Myers, P. A.; Morrison, A.; Murray, K. J. Mol. Biol. 1976, 103, 199. (37) Van Esch, J.; Hoffmann, S.; Knoll, W.; Ringsdorf, H.; Sandmann, C.; Saenger, W.; Schilken, U.; Strube, M. Manuscript in preparation.
