AIM: The final goal for performing this project is to find out the crystal structure, active binding sites and functional analysis of the domains present in the HldE protein. By finding these important factors of the protein, and making the changes in active sites can result in altering the lipopolysaccharide (LPS) layer formation in bacteria. This helps antibiotics to act more prominently towards the specific kind of problems occur in those bacterial kinds.
To achieve this, we need to perform the sequential comparison of the protein with other structurally existing proteins. Along with this, we need to purify the protein without any tags for forming crystals.
Now a days, the main problem in resolving the pathological issues is to pass the antibiotic through highly secured cell wall of the organism. The very important and the best example for this are the gram negative bacteria which has slight changes when compared to gram positive bacteria. These bacterial genes are obtaining high resistance towards the antibiotics due to the presence of an extra layer ‘Lipopolysaccharide’ (LPS) in the outer membrane(Erridge, Bennett-Guerrero et al. 2002). The inner membrane of the cell is composed of phospholipids whereas the outer layer composed with different lipids called Lipopolysaccharide’s. This LPS also known as Endotoxins(Wang and Quinn 2010) which has hydrophobic component Lipid A, and hydrophilic polysaccharides which are divided in to inner core, outer core and o antigen repeat sites(Wang and Quinn 2010). All these sites are composed of different components such as Kdo, Heptose, Galactose, Glucose and PPEtn’s. The contribution of these different compounds is shown in fig 1. Due to the presence of the extensive outer membrane formation gives more integrity, and stability to cell from different foreign invaders(Valvano, Marolda et al. 2000). For example: antibiotics and antimicrobial peptides.
The Lipid A region within the LPS made of glucosamine phospholipid(Silhavy, Kahne et al. 2010). This region can be considered as the backbone which is made up of several fatty acids(Sheu and Freese 1973), and consists an extended oligosaccharide chains which has unique repeating compounds codes towards the. This is the most toxic and hydrophobic region in LPS. This lipid A region is essential for cell growth and maintains a connection with Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) present in the inner core(De Castro, Molinaro et al. 2008). The fatty acid chain in this site helps to maintain and secure the molecule present with in the layer.
The hetero-polysaccharide chain extended from the lipid A can extend up to 10nm from the membrane. The extended saccharide chain can be useful in two different ways: 1) can help to uptake the nutrient material 2) can acts as a barriers which restricts the entry of different antibiotics and other detergent materials into the cell.
The inner core consists of heptose, mainly with 3...