| dc.description.abstract | Polyamines are ubiquitous biological cations. Their high concentration in cells, increase in rapidly growing tissues and the regulatory mechanisms that have evolved for controlling them, point to the importance of polyamines in living cells. There is evidence for direct and indirect involvement of polyamines in the regulation of enzymes. It has also been shown that spermine stabilizes ribonuclease T1 and DNA against thermal denaturation. The crystal structure of the first polyamine salt was published as early as 1949. Yet, structural studies illustrating polyamine interactions are limited. The first structure of a polyamine complexed to an organic molecule, the macrotricycle-II with cadaverine dication, was solved only in 1982. Though the structure of phenylalanine transfer RNA contained two polyamine molecules, the resolution did not permit elucidation of interaction at atomic level. In order to understand the mode of interaction of polyamines with other ubiquitous biomolecules it is necessary to determine structures of polyamines under a variety of biologically relevant chemical contexts. Towards this goal, structural studies on polyamines and their complexes with acidic amino acids were undertaken. Structures of inorganic salts of polyamines have been useful in modeling polyamine interactions and in explaining the effect of polyamines on nucleic acid conformation. The structures of the chloride salts of type NH? - (CH?)n - NH? for n = 2, 3, 4 and 6 have been determined earlier. However, repeated attempts to determine the structure when n = 5 (cadaverine), made earlier, were unsuccessful. Therefore, determination of the structure of cadaverine dihydrochloride was undertaken. Occasionally unusual polyamines are found to be present in certain organisms. It has been shown that a homolog of spermidine, symmetrical homospermidine is the major polyamine in sandal leaves. The determination of the structure of the phosphate salt of sym-homospermidine isolated from sandal leaves provided the opportunity of comparing this structure with its normal homolog.
In order to elucidate the nature of polyamine interactions, diamines of various lengths were complexed to acidic amino acids, aspartic and glutamic acids and crystallized. The following structures were determined by single crystal X-ray diffraction studies.
Putrescine complexed with:
1. L-glutamic acid
2. DL-glutamic acid
3. L-aspartic acid
Propanediamine complexed with:
4. L-glutamic acid
5. DL-glutamic acid
Hexanediamine complexed with:
6. L-glutamic acid
It has been known that certain plant viral genomes bind specific polyamines while others do not. It is possible that the information for polyamine binding is contained in the genomic sequences of these viruses. A systematic analysis of the polyamine binding and non-binding viral genomic sequences was therefore carried out.
The thesis begins with a brief description of the role of polyamines in cell function. This is followed by a description of the features of the crystal structures of inorganic salts of polyamines. During the course of these investigations structures of several polyamine nucleotide complexes became available. These structures and a few models proposed for polyamine nucleic acid interactions are discussed in context with the work reported in this thesis.
Crystallization is the first step towards successful determination of structure by X-ray diffraction. Chapter II provides the details of purification and crystallization of the structures reported in this thesis. Sym-homospermidine was purified from sandal leaves following published procedures and its phosphate salt crystallized by liquid diffusion of propanol. Free polyamines were prepared from commercially available chloride salts by ion exchange chromatography. The free amine was titrated with aspartic or glutamic acid till the pH reduced to 7. This was concentrated by lyophilisation. The aqueous concentrate was layered with a variety of non-polar solvents and left standing, to undergo liquid diffusion. Though many solvents were tried, crystals useful for diffraction studies were usually obtained with propanol.
The crystal structure determination of the chloride salt of cadaverine completes the structural investigation on diamine chloride salts as well as provides information on this unusually difficult structure. This structure along with the structure of sym-homospermidine triphosphate monohydrate are presented in Chapter III. Sym-homospermidine is a naturally occurring analog of spermidine. The phosphate group in the structure presented here is in its mono-ionic form unlike the di-ionic form present in the structure of spermidine phosphate. Both the amines have an all-trans conformation in their structures. There are two cadaverine molecules in the asymmetric unit. The arrangement of the bent cadaverine molecules in the unit cell is such that there exists a water channel with a disordered water molecule between the concave surfaces of the amine, while the space between the convex surfaces is not large enough to accommodate water molecules.
Chapter IV begins by describing the structure determination of the diamine-amino acid complexes. Packing in the crystals of the putrescine complexes are similar with the amine sandwiched between two amino acids. This type of packing allows the amines to have van der Waals type of interactions with the backbone nonpolar atoms of the amino acids. In order to achieve this, the putrescine assumes a gauche-trans-gauche conformation in the aspartic acid complex. In the other two complexes the putrescine is present in an all-trans conformation. The packing of molecules in the complexes of propanediamine with L and DL-glutamic acids are very different. The packing in the DL complex is similar to the packing in the putrescine complexes with the amine sandwiched.
Between two glutamic acid molecules of the same chirality, suggesting this could be the structure in solution as well as in its L-complex. However, the basic packing unit in the propanediamine L-glutamic acid structure are dimers of glutamic acid molecules interspersed with propanediamines. The propanediamine, in order to effectively pack in such a situation, assumes a trans-gauche conformation. The hexanediamine glutamic acid complex is the only complex in which there are water molecules of crystallization. The distance between the carboxyl groups of glutamic acid, irrespective of the conformation of the glutamic acid molecule, is shorter than the distance between the amino groups of hexanediamine in its all-trans conformation. Water molecules act as hydrogen bonding bridges between the amino groups of hexanediamine and the carboxyl groups of the amino acid.
The major aim of determining these structures was to perform a systematic analysis of the electrostatic, hydrogen bonding and van der Waals interactions in these structures and understanding their implication for the binding properties of polyamines. Procedures for these analyses were developed and applied to the complexes. The stability of these complexes depends on the contributions from all the three types of interactions. The relative strengths of these interactions in three of these complexes were quantitated. The capacity of polyamines to take part in all the three types of interactions is probably one of the reasons for their choice as biological cations.
Polyamines bind selectively to certain plant viruses. In an attempt to explain this selectivity, sequences of 30 plant viral genomes from the EMBL database were chosen and analyzed. This analysis and its results form the content of Chapter V. No consensus sequence was found in polyamine binding viruses that could be attributed to sites of binding. However, it was noticed that the departure from expected frequencies of dinucleotides was greater in polyamine binding viruses. A skewness parameter, defined to signify the deviation from the anticipated frequency, was computed. Polyamine binding viruses had the largest skewness parameters. Also, the change in frequency from anticipated values of complementary oligonucleotides was simultaneously higher or lower in these viruses. Computations were performed to understand this phenomenon. These computations establish a definite correlation between polyamine binding and double helical potential in excess of the extent anticipated for random distribution of nucleotides. This property along with the presence or absence of the basic amino terminal arm might be determinants of polyamine binding.
A summary of all the results obtained during the course of these investigations is presented in the last chapter (Chapter VI).
All the work reported in this thesis has either been published or communicated for publication. The publications based on this work are as follows:
1. Crystal structure of putrescine-glutamic acid complex. S. Ramaswamy, M. Nethaji and M. R. N. Murthy, (1989) Current Science, 58, 1160-1162.
2. Crystal structure of putrescine-aspartic acid complex. S. Ramaswamy and M. R. N. Murthy, (1990), Current Science, 59, 379-382.
3. Crystal structure of hexanediamine-glutamic acid complex. S. Ramaswamy and M. R. N. Murthy, (1991) Current Science, 60, 173-176.
4. Crystal and molecular structure of putrescine DL-glutamic acid complex. S. Ramaswamy and M. R. N. Murthy, (1991), Current Science, 61, 410-413.
5. Crystal structure of sym-homospermidine triphosphate monohydrate. S. Ramaswamy and M. R. N. Murthy. (accepted for publication in Indian J. Biophys. Biochem.).
6. Crystal structures of propanediamine complexed with L and DL-glutamic acid: effect of chirality on propanediamine. S. Ramaswamy and M. R. N. Murthy. (accepted for publication in Acta Cryst. Section C.).
7. Crystal structure of cadaverine dihydrochloride monohydrate. S. Ramaswamy and M. R. N. Murthy. (communicated).
8. Analysis of the nucleotide sequences of plant viral genomes: statistically significant occurrence of complementary nucleotides. S. Ramaswamy and M. R. N. Murthy. (communicated).
9. Polyamines: structure and interactions. S. Ramaswamy and M. R. N. Murthy, (communicated). | |
