447. Figure 8. Section of the crystal structure of the small protein crambin, Shown is a cluster composed of water pentagons. Taken from Teeter (1985).
Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 1 & 2, August 1985, pp. 437–450. © Printed in India.
Hydrogen bonding patterns and dynamics in the hydration of biological macromolecules W. SAENGER‡, Ch. BETZEL, V. ZABEL, G. M. BROWN*, B. E. HINGERTY*, B. LESYNG** and S. A. MASON † Institut für Kristallographie, Frie Universitat Berlin, Takustr. 6, 1000 Berlin 33, Federal Republic of Germany * Oak Ridge National Laboratory, Oak Ridge, Tennesse, USA ** Institute of Experimental Physics, Warsaw University, Warsaw, Poland † Institut Laue-Langevin, Grenoble, France Abstract. In crystalline cyclodextrin hydrates O–H . . . Ο hydrogen bonds occur in homodromic chainlike and cyclic motifs. In β-cyclodextrin· 11 H2O, where OH-groups are disordered, flip-flop hydrogen bonds O–(1/2H) . . . (1/2H)–O are found which represent a dynamical equilibrium O–H . . . . . . H–O. Detailed insight into otherwise hidden structural aspects of hydration and water structure (clusters) become available. Keywords. Hydrogen bonds; cyclodextrins; flip-flop dynamics; neutron crystal structure.
Introduction The function of biological macromolecules is closely associated with their structures. First of all, these structures are stabilized by covalent bonds which link individual units such as amino acids, nucleotides or sugars. The folding of the macromolecules in their specific three-dimensional native forms is achieved by the non-directional hydrophobic and van der Waals forces and by the directional hydrogen bonds. The latter are responsible for the arrangement of polypeptide chains in the form of α-helices and β-pleated sheets, and they link complementary strands in DNA by formation of Watson-Crick base pairs. Besides their importance in the stabilization of certain specific structure elements, hydrogen bonds are of particular interest in biological recognition processes and in the hydration of the biological macromolecules. Hydrogen bonds always involve hydrogen atoms which for technical reasons cannot be located by X-ray diffraction methods in macromolecular structures with molecular weights above, say, 2000 daltons. A way out of this dilemma could be the application of neutron diffraction if technical problems like low flux and limited size of crystals did not inhibit detailed investigations. In order to study hydrogen bonding patterns in larger molecules, therefore, recourse has to be made to model systems which are small enough to be studied thoroughly with X-ray and neutron diffraction methods, and which are large enough to give a picture that can be extrapolated to biological macromolecules. ‡ To whom correspondence should be addressed.
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Very small organic or biological molecules can serve as a first step in these investigations in order to derive basic principles. Crystal structure analyses of such systems will give information on individual hydrogen bonds which occur in the core of nucleic acids or proteins. If association of hydrogen bonds into more extended patterns is considered as they are observed at hydrated surfaces of macromolecules, crystal structures of larger molecules have to be analyzed. Suitable model systems for such studies are cyclodextrins which contain a large number of OH groups. They cocrystallize with water and provide new insight into the formation and dynamics of hydrogen bonding patterns. Cyclodextrin-hydrate crystal structures as a source for complex OH . . . O hydrogen bonding patterns If starch is degraded by enzymes called glucanotransferases, cyclodextrins (cycloamyloses) are obtained. They are a family of cyclic oligosaccharides composed of six, seven or eight α-D-glucopyranose units in α(1-4) linkage and called α-, β-, γ-cyclodextrins, respectively (figure 1) (Bender and Komiyama, 1978; Szejtli, 1982; Saenger, 1980). The cyclodextrins contain two secondary and one primary OH groups per glucose. Since they crystallize from water with six (α), eleven (β), and eighteen (γ) hydration water molecules, there are a large number of OH groups in a unit cell (table 1) which give rise to extended O–H . . . Ο hydrogen bonding networks. The cyclodextrin crystals diffract
Figure 1. Chemical structure of β-cyclodextrin. Oxygen atoms are indicated by filled circles, hydroxyl groups by. he insert explains the glucopyranose numbering scheme.
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Table 1. Some physical data of cyclodextrin-hydrates.
well to 0·8 Å resolution or better, so that all C, Ο atoms can be located reliably. In some cases, Η atoms were clearly seen in X-ray difference Fourier maps and for their unambiguous positioning, neutron diffraction has been employed in α-, β- and γcyclodextrin hydrate crystal structures. Hydrogen bonds are therefore well defined and patterns can be described with confidence.
Infinite hydrogen-bonding systems form chain-like and cyclic structures with preferred homodromic arrangements In α-, β- and γ-cyclodextrin crystal structures, it was found that infinite chains of O–H . . . O–H . . . O–H hydrogen bonds are formed in which all O–H . . . Ο bonds are uni-directional and called homodromic. These infinite chains can also close up to produce cycles with four, five, six or more O–H groups, see figures 2, 3 (Saenger, 1979; Saenger and Lindner, 1980). Besides the homodromic arrangement, in some cycles an anti-dromic system was observed. Here, one water molecule donates two hydrogen bonds which give rise to two chains running in opposite directions and colliding at one oxygen acting as double acceptor. The heterodromic case with no obvious order of O–H . . . Ο direction has not been observed thus far.
Figure 2. Definition of (a) homo-, (b) anti, and (c) heterodromic arrangement of hydroxyl groups. The same nomenclature is also used for infinite chains (Saenger and Lindner, 1980).
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Figure 3. Sections of the crystal structures of α-cyclodextrin-6H 2O, form I (a, neutron study) and of form II (b, X-ray study). Homo and antidromic cycles are shown by circular arrows with one and two heads respectively. Water molecules are designated by W, hydroxyl oxygens by e.g. O(6)3, where the number in parentheses describes the atom in the glucose, and the number after parentheses indicates to which of the six glucoses in the macrocycle atom O(6) belongs. Infinite, homodromic chains are marked by “chain”. (Saenger, 1979; Saenger and Lindner, 1980).
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The cooperative effect favours homodromic systems The reason for homodromic chain-like or cyclic arrangements is found in the cooperativity which results if extended to O–H . . . Ο hydrogen bonding networks (Jeffrey and Takaji, 1978; Del Bene and Pople, 1970, 1973). This cooperativity can only occur if the hydrogen bond donor acts simultaneously as acceptor, as in the case of the O–H group. If an O–H is engaged in a hydrogen bond, it is polarized such that the oxygen becomes more negative and the hydrogen becomes more positive, i.e. it is a stronger donor and acceptor compared with an isolated O–H group. For this reason, strings of O–H . . . O–H . . . O–H hydrogen bonds are preferred over single O–H . . . Ο interactions, or E(O–H . . . O)n < ∑E(O–H . . . O). n
For the cyclic and chain-like structures displayed in figure 3, the hydrogen bonding energy has been calculated using PCILO methods (Lesyng and Saenger, 1981). The results of this study are summarized in table 2. They clearly suggest that in the homodromic chain-like and cyclic arrangements, about 10% energy is gained relative to individual O–H . . . Ο hydrogen bonds. The increased strength of the interactions and the associated polarization of the O–H groups is also reflected in modifications in charge distribution and in dipole moments (Lesyng and Saenger, 1981). Table 2. Results of PCILO calculations giving stabilization energies (Kcal/mol) for hydrogen bonds in cyclic and chainlike arrangements. Cycles are I, II, III of figure 3a and IV of figure 3b and the infinite chain 1 of figure 3a. Etot is the total interaction energy, ∆Ehb represents the energy of all the hydrogen bonds forming a hydrogen-bonding system and n is the number of those hydrogen bonds. The interaction energies are always negative (Lesyng and Saenger, 1981)
a
Results are for one asymmetric unit.
Cyclic structures are often fused to form more extended patterns In many cases, the cyclic structures do not occur isolated but they are fused to form more extended patterns. Shunts or connections are provided in most cases by water
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molecules which engage their two donor sites in two different cycles. As displayed in figure 3, homo- and antidromic cycles can be fused and infinite chains can be incorporated. Similar fused cycles have already been described in the structures of different ice modifications and of the ice clathrates (Pauling, 1935; 1960; Hollander and Jeffrey, 1977). In these systems, however, high symmetry space groups require formation of cyclic arrangements. In contrast, in the cyclodextrin crystal structures, cyclic motifs occur because they represent favourable structures if many O–H groups and water molecules associate. Flip-flop O–H . . . H–O hydrogen bonds in β-cyclodextrin In the β-cyclodextrin 11H2O crystal structure, the eleven water molecules are distributed statistically over 16 sites (Betzel et al., 1985). The distribution is not even, some sites being fully occupied and some others to only 20 %. Hydrogen atom positions were determined by neutron diffraction methods. In this crystal structure, besides normal O–H . . . Ο hydrogen bonds, a large number of O–H . . . H–O interactions are found. In these, the Ο . . . Ο distances are in the usual range, 2·7–2·9 Å, and hydrogen atoms are only ~ 1 Å apart, i.e. shorter than the van der Waals Η . . . Η distance of 2·4 Å. Consequently, they cannot be present simultaneously; their positions are occupied only ~ 0·5 and in each O–H . . . H–O, hydrogen atom occupancies add up to ~ 1·0. Because these hydrogen bonds represent a Statistical average over two states
they were termed flipflops (Saenger et al., 1982). In the β-cyclodextrin crystal structure, several of these flip-flop hydrogen bonds form more extended systems, (see figure 4) all of which can occur in two states. If transition from one to the other state takes place, all hydrogen bonds in a system have to rotate from one into the other position in a concerted, cooperative motion.
Are flip-flops static or dynamic? The occurrence of O–H . . . H–O bonds can be due either to a dynamic disorder as described above or to a static disorder. In the latter, some domains in the crystal or some unit cells in the structure are in one state, say O–H . . . O, and in other domains or unit cells, in the other state Ο . . . H–O. This static disorder should be independent of temperature whereas dynamic disorder should disappear if cooled down. In order to differentiate between these two possibilities, a neutron diffraction study was carried out at 120K (Zabel et al., 1975). It clearly demonstrated that most of the flip-flop hydrogen bonds disappear, (figure 5), and a quadrilateral cycle was newly formed (figure 6) i.e. the flip-flops represent a dynamic equilibrium in the solid state.
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Figure 4. (a) Section of a Fourier difference map calculated from neutron diffraction data for β−cyclodextrin-11H2O, showing D atom positions (referred to as Η atoms in the text). Flipflop hydrogen bonds O–H . . . H–O are indicated by stippling. (z), Positions of Η, Ο and C atoms–––, covalent bonds; – – –, hydrogen bonds. (b) Same section as in (a), giving an interpretation at the atomic level and showing the deconvolution of the flip-flop hydrogen bonding system into two states (I and II) having normal hydrogen bonds of the type O–H . . . O. Solid arrows indicate the manner in which O–H bonds may rotate in concerted, cooperative motion from one state to another. Circles of increasing size represent H, C and Ο atoms, with C shown shaded and Η filled. Covalent bonds are indicated by solid lines, Η bonds by open lines. The water molecule, W2, displays three Η atom positions, one (C) fully occupied with an occupancy factor 0·99(1) whereas A and Β are only partly filled, 0·44(1) and 0·49(1) (Saenger et al., 1982).
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Figure 5. In the β−cyclodextrin-11H2O crystal structure, all intramolecular, interglucose hydrogen bonds are of the flip-flop type. Hydroxyl hydrogens are drawn black. If positions A are filled, position Β have to be empty and vice versa. (Betzel et al., 1985).
Flip-flops occur in β-cyclodextrin, in infinite chains and in cycles In the cyclodextrins, all O(2) and O(3) hydroxyls are on the same side of the macrocyclic molecule and are able to form intramolecular, interglucose Ο(2) . . . O(3) hydrogen bonds. In the case of the β-cyclodextrin-11H2O crystal structure, all of the seven intramolecular hydrogen bonds are of the flip-flop type, (figure 5). They are probably responsible for the rigidity of the molecule in aqueous solution, which is reflected in circular dichroism (CD) spectra and in H/D exchange experiments (Saenger, 1980). In the crystal structure determined at 120K, all but one of these flip-flops disappear, (figure 6). There is one infinite flip-flop chain (. . . H–O–H . . . H–O–H . . . H–O . . . )n run-
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Figure 6. At 120K, the flip-flops disappear in the β-cyclodextrin-11H2Ο crystal structure because they are due to dynamic, and not static disorder. However, a new quadrilateral, all flipflop cycle is formed. Taken from Zabel et al., (1985).
ning through the crystal structure at room temperature. If the crystal is cooled to 120K, this chain transforms into a chain with normal O–H . . . Ο hydrogen bonds in a homodromic arrangement (figure 7). At 120K, a quadrilateral flip-flop cycle is formed which does not exist at room temperature (figure 6). If the crystal is heated up again, the cycle disappears. This arrangement obviously represents an energetically preferred situation at low temperature, but at room temperature, another hydrogen-bonding situation is energetically more favoured. Flip-flops in the structures of ice and ice clathrates Comparable flip-flop motifs like the quadrilateral cycle and the infinite chain in the crystal structure of β-cyclodextrin-11H2O are found in the structures of ice and ice clathrates. In these, however, the flip-flop disorder has to occur because it is required by space group symmetry. In contrast to the β-cyclodextrin-11H2Ο crystal structure, the disorder remains even if ice is cooled down and gives rise to a residual entropy of 0·83eu (Pauling, 1935; 1960).
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Figure 7. An infinite flip-flop chain running through the crystal structure in β-cyclodextrin11H2O. At 120K, it changes to a homodromic chain with all O–H groups pointing in the same direction.
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Figure 8. Section of the crystal structure of the small protein crambin, Shown is a cluster composed of water pentagons. Taken from Teeter (1985).
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Cycles and flip-flops—a dynamical picture of the structure of water According to the flickering cluster theory of Frank and Wen (1957), water is structured and consists of water molecules associated in dynamically changing ice-like structures which are embedded in bulk water. Based on the concept of fused cyclic arrangements of water molecules with four-, five-, and six-membered rings predominating as found in the crystal structures of the cyclodextrins, we can envisage that combination with the cooperative effect and flip-flop dynamics would create very flexible “flickering clusters”. They could vary rapidly with time in size and in structure and produce even a picture
Figure 9. Water (pentagon) structure observed in the dideoxynucleosidephosphateproflavin complex. Atoms of the nucleotide and drug molecules are omitted for clarity. Neidle et al. (1980).
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where water as a whole is “flickering”, i.e. the distinction between “flickering clusters” and “bulk water” becomes meaningless.
Hydration in proteins and nucleic acids displays pentagon motifs The recently published high-resolution crystal structures of a small protein, crambin (Teeter, 1985; figure 8), of a dideoxynucleoside-phosphate-proflavin complex (Neidle et al., 1980; figure 9), and of an octanucleotide duplex (Kennard, 1984; figure 10) display surface-bound water in a certain, nonrandom arrangement. The water molecules form pentagons where all oxygen atoms are in hydrogen-bond distance. The pentagons are fused to produce more extended patterns, similar to those observed for the cyclodextrin-hydrate and for the ice clathrate crystal structures. Because hydrogen atoms could not be located in these large molecule crystal structures, details of the hydrogen bonds are still not clear. For comparison with the β-cyclodextrin-11H2O crystal structure, it would be of interest to know whether they are of the O–H . . . Ο or of the flip-flop O–H . . . H–O type. In any case, the hydrogen-bonding (or watermolecule) motifs observed at the surface of these biological macromolecules is so close
Figure 10. Structure of an octanucleotide duplex occurring in the A-DNA form. In the major groove, water molecules are arranged in pentagons outlined by shading. Kennard (1984).
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to those found in the crystal structures of cyclodextrin hydrates that the analogy is clear. Further studies, therefore, are encouraged and more model systems should be investigated thoroughly so that we are able to understand the complex situations occurring in biological macromolecules. Acknowledgements This work was supported by Bundesministerium für Forschung und Technologie (FKZ O3B72A79), by Deutsche Forschungsgemeinschaft (Sa 196/11-1) and by Fonds der Chemischen Industrie. G.M.B, and B.E.H. were sponsored by Division of Materials Sciences, Office of Basis Energy Sciences, US. Dept. of Energy and by Office of Health and Environmental Research, US. Dept. of Energy, respectively, under contract DEACO5-84OR21400 with Martin Marietta Energy Systems, Inc. References Bender, Μ. L. and Komiyama, Μ. (1978) “Cyclodextrin Chemistry”, (SpringerVerlag) Betzel, Ch., Saenger, W., Hingerty, B. and Brown, G. M. (1985) J. Am. Chem. Soc. (in press). Del Bene, J. Ε. and Pople, J. Α. (1970) J. Chem. Phys., 52, 4858. Del Bene, J. E. and Pople, J. A. (1973) J. Chem. Phys., 58, 3605. Frank, Η. S. and Wen, W. Y. (1957) Discuss. Faraday Soc., 24, 133. Hollander, F. and Jeffrey, G. A. (1977) J. Chem. Phys., 66, 4699. Jeffrey, G. A. and Takaji, S. (1978) Acc. Chem. Res., 11, 264. Kennard, O. (1984) Pure Appl. Chem., 56, 989. Lesyng, B. and Saenger, W. (1981) Biochim. Biophys. Acta, 678, 408. Neidle, S., Berman, H. M. and Shieh, H. S. (1980) Nature (London), 288, 129. Pauling, L. (1935) J. Am. Chem. Soc., 57, 2680. Pauling, L. (1960) “The Nature of the Chemical Bond”, 3rd edn, (Cornell Univ. Press) p. 467. Saenger, W. (1979) Nature (London), 279, 343. Saenger, W. (1980) Angew. Chem. Int. Ed. Engl., 19, 344. Saenger, W., Betzel, Ch., Hingerty, Β. and Brown, G. Μ. (1982) Nature (London), 296, 581. Saenger, W. and Lindner, K. (1980) Angew. Chem. Int. Ed. Engl., 19, 383. Szejtli, J. (1982) Cyclodextrins and their inclusion complexes Akademiai Kiado, Budapest. Teeter, M. M. (1985) Proc. Natl. Acad. Sci. USA, (in press) Zabel, V., Saenger, W. and Mason, S. A. (1985) J. Am. Chem. Soc., (in press)
