X-ray studies on some crystalline complexes between amino acids with ionisable sile chains

Abstract

The structure, assembly, and function of proteins critically depend upon different types of non?covalent interactions involving amino acid residues. In fact, it is increasingly clear that the extreme subtlety and high complexity of biological systems depend largely upon the presence of such comparatively weak interactions. Though protein crystallographic studies have provided a wealth of information on these interactions, their atomic details cannot be understood solely from such studies on account of the limited resolution of most protein electron?density maps. Therefore, a programme of X?ray investigations on crystalline complexes involving amino acids and short peptides has been undertaken in an attempt to study, at atomic resolution, the geometric features of the possible non?covalent interactions among them. These investigations can also contribute to the understanding of the structure and conformation of amino acids and their residues. The work carried out by the author as part of the above programme on some crystalline complexes between amino acids with ionisable side?chains is reported in this thesis. As is well known, lysine, arginine, histidine, aspartic acid, and glutamic acid have readily ionisable side?chains. An analysis of the structure and conformation of these side?chains observed in crystal structures, including those of globular proteins, is given in the first chapter. The average bond lengths and valence angles in the side?chains have been derived from the crystal structures of the five amino acids, their derivatives, and salts. The side?chain conformations observed in these crystal structures as well as in those of the metal–amino acid complexes have been discussed in detail. A statistical analysis of the conformation of these side?chains in globular proteins is also presented in this chapter. Chapter 2 deals with the preparation and preliminary X?ray studies of the crystalline complexes. Taking one basic amino acid and one acidic amino acid at a time, a total of six binary complexes between amino acids with ionisable side?chains are possible. Of these, crystals (suitable for X?ray analysis) of only three—namely, L?lysine L?aspartate, L?arginine L?glutamate monohydrate, and L?histidine L?aspartic acid monohydrate—could be grown. The unit?cell dimensions and the space groups of the crystals were determined from oscillation, Weissenberg, and precession photographs. The X?ray analysis of L?lysine L?aspartate is reported in Chapter 3. The structure (space group P21P2_1P21?; Z=2Z = 2Z=2) was solved by direct methods and Fourier techniques and refined to an R value of 0.068 for 1134 photographically observed reflections. The dimensions and conformation of the lysine molecule are similar to those observed in lysine monohydrochloride dihydrate. The side?chain carboxyl group of the aspartate ion is deprotonated, unlike that in L? and DL?aspartic acid. The bond lengths and angles in the aspartate ion, except those in the side?chain carboxylate group, are comparable with those in L? and DL?aspartic acid. Some differences, however, exist in the conformation of the molecule in these structures. The crystal structure of lysine aspartate consists of alternating layers—one layer comprising lysine molecules and the other, aspartate ions. The two layers are interconnected primarily by hydrogen bonds between the side?chain amino group of lysine and carboxylate oxygen atoms belonging to neighbouring aspartate ions. Chapter 4 reports the crystal structure of L?arginine L?glutamate monohydrate. The structure (space group P212121P2_12_12_1P21?21?21?; Z=4Z = 4Z=4) was solved by the symbolic?addition procedure and refined to an R value of 0.083 for 1007 photographically observed reflections. The conformation of the arginine molecules in this structure is different from those reported so far, whereas the conformation of the glutamate ion is the same as that observed in the structure of glutamic acid hydrochloride. As in the case of lysine aspartate, the crystal structure consists of alternating layers—one layer consisting of cationic arginine molecules and the other containing negatively charged glutamate ions. The adjacent layers are interconnected principally through a specific ion?pair interaction between the guanidinium group of arginine and the ??carboxylate group of glutamate, and a water bridge. The former, which involves the electrostatic attraction between the positively charged guanidinium group and the negatively charged carboxylate group as well as two nearly parallel N–H···O hydrogen bonds, is the first such specific side?chain–side?chain interaction to be observed at atomic resolution. The final chapter deals with the structure of L?histidine L?aspartic acid monohydrate. The X?ray analysis was complicated by the presence of (i) non?crystallographic screw axes, (ii) a pseudo?translation of c/2c/2c/2, and (iii) twinning. The structure solution was achieved in several stages following a rather tortuous path. The diffraction pattern falsely indicated the space group to be P21212P2_12_12P21?21?2, Z=8Z = 8Z=8, with the two sets of crystallographically independent and chemically identical molecules related to each other by a pseudo?translation c/2c/2c/2. In the course of the analysis, it was discovered that the crystals were monoclinic (space group P21P2_1P21?; ?=90?\beta = 90^\circ?=90?; Z=8Z = 8Z=8) and were twinned about the aaa or the bbb axis. The crystallographic asymmetric unit thus contains four molecules each of histidine, aspartic acid, and water. Half of these are related to the other half by non?crystallographic 212_121? screws parallel to the aaa axis. In each half, the two aspartic acid molecules (and the two water molecules) are related to each other by a pseudo?translation c/2c/2c/2, whereas the two histidine molecules are not. The twinned structure was refined to an R value of 0.113 by a modified structure?factor least?squares procedure. All the aspartic acid molecules in each twin component have essentially the same—and a rather unusual—conformation, with the side?chain carboxyl group gauche to both the ?\alpha??amino and the ?\alpha??carboxyl groups. Half the histidine molecules in the structure exist in an open conformation, whereas the other half exist in a closed conformation. The arrangement of molecules in the structure is similar to that in lysine aspartate and arginine glutamate monohydrate, in that the basic and acidic amino acids first organise themselves into two?dimensional aggregates, which are then packed together in an alternating fashion to form the crystal. The present structure, however, consists of alternating double?layers—one double?layer made up of histidine molecules and the other of aspartic acid molecules. The water molecules are sandwiched between the two layers in the aspartic acid double?layer. The adjacent double?layers are interconnected by hydrogen bonds between the amino and imidazole groups of histidine molecules on the one hand and the carboxyl groups of the aspartic acid molecules on the other.