STRUCTURAL STUDIES ON 11S GLOBULIN AND 2S ALBUMIN FROM WRIGHTIA TINCTORIA

Abstract

From the human point of view, the most important part of plant is seed that is harvested and consumed in countless forms. The seed storage protein constitutes an indispensible dietary protein that account 70% of the total intake. In addition, these proteins are sole source for the non ruminating farm animals. The storage proteins constitute a significant part of plant seeds that governs several inherent traits such as viability, nutrient value, protection from microbe and insects as well as end use properties. Although, diverse kinds of proteins are present in seed that confer metabolic and structural support, nevertheless almost every seed contains some proportion of the storage proteins. They are mostly present in high amount and facilitate store of amino acids, consumed during seed germination and seedling growth. The specific quality and texture of seed is determined by property of storage protein. In particular example of cereals, the lysine, tryptophan and threonine are limited, while in legumes methionine is the deficient amino acids. This deficiency is due to their limited proportions in seed storage proteins. The classification of seed storage proteins (SSPs) is primarily based on the medium of solubility and extraction, the proteins are soluble in, i) dilute saline classified as globulins, ii) water classified as albumins, iii) alcohol classified as prolamin and iv) dilute acid/alkali classified as glutelins. The globulins are most profoundly distributed form of seed storage protein marks its presence across dicots, monocots as well as in lower forms of plant such as ferns. Globulins are large globular proteins further graded in two groups based on differences of sedimentation coefficients, as 11S legumin types and 7S vicilin types. The post translational processing confers major difference among these classes, which is visible in form of difference in structural organization and post translational modifications. Although both of proteins are deficient for cystine and methionine, 11S globulins are relatively higher in sulfur containing amino acid than 7S class. The 11S globulin is almost always found together with 2S albumin. The 2S albumins are smallest member of the SSP family. They are classified on the basis of sedimentation coefficient. The 2S albumins are widely known for their conserved and higher sulphur containing amino acids property. The substantial proportion of sulphur containing amino acids make them most suitable SSPs that can be exploited for improvement of seed nutrition and also for other related applications where sulphur content is required. In addition to that the high level of cystine in 2S albumins enables them to acts as a weapon in plant defence II system. A less studied regulatory role of calmodulin antagonist function has been also found from the 2S class of seed storage protein. This thesis is focused on structural and functional studies of 11S globulin and 2S albumin from the plant Wrightia tinctoria. The comprehensive structure analysis and the related functions have been described in four sections of chapters. An independent work has been also incorporated under the section appendices. That contains crystallization study for structural analysis of a penicillin binding protein FmtA, responsible to confers antibiotic methicillin resistance to the gram positive bacteria S. aureus. Chapter 1. reviews the state of art in literature; describes structural fictional details of 11S globulin and 2S albumins. Chapter 2. describes the identification, purification and biochemical characterizations of 11S globulin from the plant W. tinctoria. An 11S class of seed storage protein has been purified and the relation of the protein to 11S family has been established by N-terminal as well as partial sequencing and further the gene responsible for their synthesis was cloned. The basic molecular profile has been revealed that describes the W. tinctoria globulin (WTG) as a hexamer made up of six monomers and further each monomers are constituted by two different alpha and beta subunits attached together by disulphide bond. It has been observed that WTG naturally exists in a very polymorphic and heterogeneous form of their constituent subunit components, as observed from internal sequencing as well SDS-PAGE analysis. The cloning of entire gene and the corresponding amino acid profile reveals that WTG is made up of 482 amino acid residues. Furthermore, the biochemical characterizations reveal that WTG encompasses a unique ability of hemagglutination. It has been observed that WTG also contains exceptional stability, concluded on the basis of circular dichroism and fluorescence studies. Only the high concentration and harsh ionic condition can sufficiently change the native state, otherwise WTG can tolerate wide range of chaotropic reagents and pH conditions. Chapter 3. focuses on the structural studies of WTG by crystallization and structure determination. The X-ray diffraction studies have been employed for elucidation of structural III and functional details. With a high diffraction up to 1.8 Å resolutions, the data has been solved and the final model was build. The refined model is showing all the crystallographic parameters in defined limits. The WTG encompasses conserved structural fold of jelly roll β-barrel motif that constitute a bicupin fold as a signature of cupin super family. Furthermore, the extended alpha helical domains along with cupin fold draw the structural analogy with members of 11S SSPs. The WTG encompasses six monomers, which constitute a hexameric scaffold. Further, WTG structure showed that there are three fold symmetry exists between two trimers and this view depicts a trimer of dimer model of WTG, along perpendicular to three fold a twofold symmetry has been also observed for WTG but this symmetry showed dimer of trimer arrangement of WTG. The N-terminal regions of both trimers remain funnel together at the centre of three fold axis at two opposite ends; this arrangement constitute the two poles of WTG hexamer. The initial 20 residues of N-terminal region are showing exception hydrophilic nature as well as presence of serine residue having fairly high score for possible phosphorylation. Hence it appears form the WTG model that there is a delicate series of interaction involved in hexamer formation. In addition to that dimer of trimer or trimer of dimer, both view forms three pockets related along three fold axis of symmetry. The high resolution data clearly revealed an auxin molecule uniformly present in three pockets of hexamer. Therefore, it appears that a mild change of pH or alteration of surrounding environment WTG may lead to dissociation of entire hexamer assembly. Chapter. 4. describes identification, purification, biochemical and biophysical characterizations of W. tinctoria 2S albumin (WTA). The WTA was purified to homogeneity in three steps of purification. The purified WTA was subjected to partial internal sequencing and obtained sequence were aligned to confirms the relation with albumin family of protein from the W. tinctoria against available EST database of NCBI. The basic secondary structure profile of WTA has been revealed by circular dichroism spectral analysis, which concludes it as a major alpha helical protein. The highly conserved cystine residues and substantial ionic surface of WTA have been speculated for metal binding activity; hence the biochemical interaction has been studied against different metals by employing isothermal titration calorimetory. The ITC results confirm a significant binding of WTA with Copper, Cadmium and Lead. In addition to this the in silico modeling and structure analysis reveal that WTA contains reasonable hydrophilic surface profile that also supports the metal binding. IV Appendix A. describes the purification and crystallization studies of FmtA from S. aureus. Review and related literature has been surveyed in the chapter. For the structural studies of FmtA, the protein was purified in two steps involving ion exchange and gel filtration chromatography. The purified protein was subjected to crystallization trial over 96 well sitting drop trays, and crystals were obtained. Initial diffraction data showed that the model is following the molecular weight relation to the cell volume correlation, as well the densities are coming closely similar to the related homologues and matching the sequence profile with two molecules in asymmetric unit. The more organized crystals are under optimization for the FmtA structure solution and to building of final model.