X-ray crystallagraphic studies on the effect of chirality in amino acids aggregation

Abstract

A programme of systematic X-ray studies on crystalline complexes involving amino acids and peptides is currently under way in this laboratory. These studies, apart from providing a wealth of information on non-covalent interactions which play a crucial role in the structure, function and assembly of proteins, have resulted in a detailed understanding of different well-defined patterns of amino acid and peptide aggregation. In particular, it has been shown that head-to-tail sequences, in which the ?- or terminal amino and carboxylate groups are brought into periodic hydrogen-bonded proximity in a polypeptide-like arrangement, are an almost universal feature of amino acid and peptide aggregation. These sequences assume characteristic geometries and could form the basis for predicting crystalline patterns. This thesis is concerned with the author’s contribution to the ongoing programme on crystalline complexes. The current focus of this programme has been the possible relevance of molecular interactions and aggregation to chemical evolution. The same is true about the work reported in this thesis. A brief description of the previous work pertaining to this particular aspect is therefore given in the introductory chapter. This thesis is particularly concerned with the effect of chirality on amino acid aggregation and its possible relevance to chemical evolution. Although the X-ray analysis of two binary complexes containing amino acids of mixed chirality, namely L-arginine D-glutamate trihydrate and L-arginine D-aspartate, has provided interesting insights into the effect of chirality on molecular aggregation, more complexes involving D as well as L amino acids need to be analysed before firm conclusions can be drawn regarding chiral effects. Therefore, the author has taken up a detailed study of crystalline complexes involving amino acids of mixed chirality. A crucial, and at times difficult, step in the X-ray studies on crystalline complexes is the preparation of suitable single crystals. Chapter 2 describes the crystallisation experiments-unsuccessful as well as successful-carried out by the author. Crystals of the following complexes suitable for X-ray studies could be grown: 1. L-lysine D-glutamate 2. L-lysine D-aspartate 3. L-ornithine D-aspartate 4. A new form of L-arginine D-glutamate 5. DL-arginine DL-glutamate 6. DL-arginine DL-aspartate 7. DL-arginine acetate 8. DL-lysine acetate The crystal structures of two LD complexes involving lysine are reported in Chapter 3. The lysine molecule in L-lysine D-glutamate assumes a conformation with the side chain staggered between the ?-amino and the ?-carboxylate groups. The molecular aggregation is very similar to that in L-arginine D-aspartate and L-arginine D-glutamate trihydrate. The molecules arrange themselves as double layers. The core of each double layer consists of two parallel sheets; each involving both types of molecules. The hydrogen bonds within each sheet and those that connect the two sheets give rise to LL, DD and DL type head-to-tail sequences. Adjacent double layers are held together by side chain–side chain interactions. In contrast, the unlike molecules in L-lysine D-aspartate monohydrate aggregate into separate alternating layers as in the case of most LL complexes. The arrangement of molecules in the lysine layer is nearly the same as that in L-lysine L-aspartate, with head-to-tail sequences as the central feature. The arrangement of aspartate ions in the layers containing them is, however, unusual. The aspartate ions form hydrogen-bonded helical columns around crystallographic 2? screw axes. Adjacent aspartate columns are connected through a column of water molecules. The aspartate ions do not take part in head-to-tail sequences. The interactions of the side chain amino groups of lysine in the two complexes are such that they form infinite sequences containing alternating amino and carboxylate groups. The crystal structure of L-ornithine D-aspartate monohydrate is presented in Chapter 4. The aggregation pattern in it is entirely different from that in L-ornithine L-aspartate hemihydrate. It is similar to that in L-lysine D-aspartate monohydrate in that the unlike molecules aggregate into separate alternating layers, but is different from that in the other three LD complexes. The organisation of molecules in the ornithine layer is similar to that in the lysine layer in L-lysine D-aspartate monohydrate, with two head-to-tail sequences stabilising the layer. The arrangement of the aspartate ions, however, is very different from any observed so far in crystal structures containing aspartic acid or aspartate ions. The aspartate layer is made up of molecular ribbons, each containing one head-to-tail sequence. Adjacent ribbons are interconnected through water molecules and side chain amino groups. An interesting feature of the structure is an internal water bridge between the ?-amino group and one of the side chain carboxyl oxygen atoms of the aspartate ion. The structure contains a closed hydrogen-bonded loop made up of alternating amino and carboxylate groups. The structure of the new form of L-arginine D-glutamate is reported in Chapter 5. In terms of composition, the new form differs from the old form in that the former is a monohydrate whereas the latter is a trihydrate. The conformation of the arginine molecule is the same in both the forms, whereas that of the glutamate ion is different. The change in the conformation of the glutamate ion is such that it facilitates extensive pseudosymmetry in the new form. The molecules arrange themselves in double layers stabilised by head-to-tail sequences in both the forms. However, considerable differences exist between the two forms in the interface, consisting of side chains and water molecules, between double layers. A comparative study of the relationship between the crystal structures of L and DL amino acids on the one hand and that between the structures of LL and LD amino acid–amino acid complexes on the other is presented. The second part of Chapter 5: The crystal structures of most hydrophobic amino acids are made up of double layers, and those of most hydrophilic amino acids contain single layers, irrespective of the chiralities of the amino acids involved. In most cases, the molecules tend to appropriately rearrange themselves to preserve the broad features of aggregation patterns when the chirality of half the molecules is reversed, as in the structures of DL amino acids. The basic elements of aggregation in the LL and the LD complexes are similar to those found in the crystals of L and DL amino acids. However, the differences between the LL and the LD complexes in the distribution of these elements are more pronounced than those between the distributions in the structures of L and DL amino acids. Having analysed several LD complexes, it was of obvious interest to examine the corresponding DL-DL complexes. In Chapter 6, the crystal structures of DL-arginine DL-glutamate monohydrate and DL-arginine DL-aspartate, the first DL-DL amino acid–amino acid complexes to be prepared and X-ray analysed, are discussed. The aggregation in the DL-DL complexes can be described in terms of two-dimensional patterns, but unlike in the case of most LL and LD complexes, these patterns do not involve head-to-tail sequences. The basic element of aggregation in both the structures is an infinite chain made up of pairs of molecules. Each pair, consisting of an L and a D isomer, is stabilised by two centrosymmetrically or nearly centrosymmetrically related hydrogen bonds involving the ?-amino and the ?-carboxylate groups. Adjacent pairs in the chain are then connected by specific guanidyl–carboxylate interactions. The infinite chains are interconnected through hydrogen bonds to form molecular sheets. The sheets are then stacked along the shortest cell translation. The interactions between sheets involve two head-to-tail sequences in the glutamate complex and one such sequence in the aspartate complex. Thus, fundamental differences exist among the aggregation patterns in the LL, the LD and the DL-DL complexes. The differences are such that if condensation were to take place in the different aggregates, the LL aggregate is more likely to yield clean peptide fragments than the other two. Acetic acid is one of the compounds produced in simulated prebiotic synthetic experiments, and a study of its interactions with amino acids is of interest in relation to chemical evolution. The crystal structures of DL-arginine acetate monohydrate and DL-lysine acetate are reported in Chapter 7. A comparison of these structures with the corresponding L-amino acid–acetate complexes that have already been analysed could provide insights into the effect of chirality on molecular aggregation. The basic elements of aggregation in both the structures are pairs of amino acid molecules, each pair stabilised by two centrosymmetrically related hydrogen bonds involving ?-amino and ?-carboxylate groups, stacked along the shortest dimension to form columns. The pairs are held together in each column by head-to-tail sequences. The columns stack along a crystallographic axis to form layers. Adjacent layers are bridged by acetate ions. The amino acid–acetate interactions are primarily through side chains and involve specific interactions and characteristic interaction patterns. The gross features of molecular aggregation are nearly the same in DL-arginine acetate and L-arginine acetate, whereas they are substantially different in the lysine complexes. As in the L-amino acid–acetate complexes, electrostatic effects are modulated by other factors to give rise to head-to-tail sequences in the present structures also. In both cases, one of the two head-to-tail sequences in the L complex is replaced by a hydrogen-bonded loop involving ?-amino and ?-carboxylate groups in the DL complex. This may have implications for prebiotic condensation during chemical evolution. Having established the ubiquity of head-to-tail sequences in the solid-state aggregation of unprotected peptides containing common naturally occurring amino acids, it was of interest to examine if such sequences occur in peptides containing ?-aminoisobutyric acid (Aib). Aib is found in simulated experiments on prebiotic synthesis, and it is highly conformationally restrictive. The crystal structure analysis of tri-?-aminoisobutyric acid dihydrate was therefore taken up and is reported in the Appendix. The bond angles in the central residue of the tripeptide exhibit conformation-dependent asymmetry about C?. The conformation of the molecule corresponds to an incipient type II? (or type II) ?-turn. The structure contains an internal water bridge, involving two water molecules, which connects the terminal amino group and the carboxyl group of the second residue. Despite the folded nature of the molecule and the presence of water molecules, the molecules indeed form a head-to-tail sequence in the crystals. It is of the zigzag type centred around a 2? screw axis. The work described in this thesis has already been reported in the following publications:

Incipient type II ?-turn, internal water bridge and head-to-tail sequence in the structure of tri-?-aminoisobutyric acid dihydrate. Acta Cryst. (1987) C43, 1618–1621 (with C.G. Suresh and M. Vijayan). X-ray studies on crystalline complexes involving amino acids and peptides. Part XV. Crystal structures of L-lysine D-glutamate and L-lysine D-aspartate monohydrate and the effect of chirality on molecular aggregation. Int. J. Pept. Protein Res. (1988) 32, 352–360 (with C.G. Suresh and M. Vijayan). X-ray studies on crystalline complexes involving amino acids and peptides. Part XVI. Crystal structure of L-ornithine D-aspartate monohydrate. Acta Cryst. (1988) C44, 1794–1797 (with M. Vijayan). X-ray studies on crystalline complexes involving amino acids and peptides. Part XVIII. Crystal structure of a new form of L-arginine D-glutamate and a comparative study of crystal structures containing molecules of opposite chirality. J. Biosci. (1989) 14, 111–124. X-ray studies on crystalline complexes involving amino acids and peptides. Part XIX. Crystal structures of DL-arginine acetate monohydrate and DL-lysine acetate and comparison with corresponding L-amino acid complexes. J. Biomol. Struct. Dynamics (1989) 7, 269–277 (with T. Rao, R. Radhakrishnan and M. Vijayan). X-ray studies on crystalline complexes involving amino acids and peptides. Part XVII. Chirality and molecular aggregation: The crystal structures of DL-arginine DL-glutamate monohydrate and DL-arginine DL-aspartate. Biopolymers (1990) 29, 533–542 (with M. Vijayan, B. Ramakrishnan and T.N. Guru Row).