M N Ponnuswamy: Macromolecular Crystallography

Macromolecular Crystallography

1. Purification, Crystallization and X-ray structure analysis of Seed storage proteins, Cocosin and Glycinin:

               Plants store proteins in embryo and vegetative cells to provide carbon, nitrogen, and sulfur resources for subsequent growth and development. The storage and mobilization cycles of amino acids that compose these proteins are critical to the life cycle of plants. Mechanisms for protein storage and mobilization serve many different developmental and physiological functions; for example, stored protein serve as building blocks for rapid growth upon seed and pollen germination. Storage proteins are a group that comprises proteins generated mainly during seed production and are stored in the seed that serve as nitrogen sources for the developing embryo during germination.

               Seed proteins are usually synthesized as precursors having a leader prepeptide sequence and prosequence which contain the signal for transport, processing and targeting from the site of synthesis to the storage organelles. All storage proteins are synthesized on the rough Endoplasmic Reticulum and after cotranslational cleavage of the signal peptide, the polypeptides are subjected to glycosylation. The Protein disulphide isomerase involves in the disulphide formation with the help of chaperones and targeted to protein bodies.

               There are two major types of storage proteins, namely vicilins and legumins, which are distinguishable by their sedimentation coefficient (7S/11S), oligomeric organization (trimeric/hexameric) and polypeptide chain structure (single chain/disulphide-linked pair of chains). 11-S globulins are non-glycosylated proteins, which form hexameric structures. Each of the subunits in the hexamer is itself composed of an acidic and a basic chain derived from a single precursor and linked by a disulphide bond.

Coconut Globulin Cocosin from Cocos nucifera

Glycinin from Arachis hypogaea (Peanut)

2. Crystal structure determination of haemoglobin from donkey (Equus asinus):

Haemoglobin acts as an important protein for oxygen carrier in all living beings. It is a tetrameric protein that carries oxygen from the lungs to the tissues and carbon dioxide from the tissues back to lungs. The active site in each subunit consists of an iron atom, bound to four pyrrole nitrogen atoms of a porphyrin and to Ne of His (F8), with the reversibly bound oxygen occupying the sixth co-ordination position at the iron. The amino-acid identity between donkey and horse haemoglobin is 98% for alpha-subunit and the beta-subunit is conserved in all equus species. We initiated the three dimensional structure of donkey haemoglobin to study the quaternary structure-function relationship and to compare it with horse haemoglobin.