Structural And Functional Analysis: Sialic Acid

BIOCHEMISTRY III TERM PAPER

-Malvika Bakshi 14IMSILS023

Sialic acid is a part of the 9 carbon sugar acids commonly found on the cell surface glycans of mammals and also in some pathogenic bacteria. Some bacteria use sialic acid as nutrition while some use it as a way of immune evasion from the host. Since many bacteria utilize sialic acid by scavenging them from the environment, these bacteria have evolved to have dedicated transporters. The authors in this paper performed structural and functional analysis on one such sialic acid transporter (SiaT) present in a gram negative strain of bacteria(Proteus mirabilis) which uses Na+ gradient and is part of the sodium solute symporter family (SSS), this SiaT forms a complex with N-acetylneuraminic

A calorimeter consists of two cells, a sample cell in which the ligand is titrated and a reference cell which contains the buffer are surrounded by an adiabatic jacket. Each cell has a heater which controls the temperature. During an experiment, before the ligand is titrated in to the sample/receptor cell with the help of a syringe in number of aliquots, a source of constant power is supplied to the reference cell, this activates the heater present on the sample cell. As the molecules bind heat is either absorbed or released, when heat is released i.e an exothermic reaction the feedback power causes the temperature in the sample cell to decrease so as to maintain an equal temperature between the two cells and vice versa during an endothermic reaction. The raw data is the power difference between the two cells during each injection/ titration to keep the temperature between at equilibrium. Each peak in the raw data corresponds to a ligand injection, the peaks start with the largest as there are many binding sites to fill as the number of injections progress the binding sites decrease and so does the change in heat. At the end of the experiment we get a graph consisting of the binding curve formed by plotting each heat spike during each injection. These peaks are then

It’s typically used to detect changes in hydration shells which occur due to changes in size, shape or overall conformation of a molecule. These types of changes allow measurement of binding affinities of nucleic acids, proteins and micromolecules along with enzymatic activities. This method is based on the principle of thermophoresis which refers to movement of molecules along a temperature gradient. The change in temperature in a microscopic scale is created with the help of an IR- Laser within a glass capillary. The capillary contains fluorescently labeled molecules which are distributed evenly in the solution, as soon as the IR-Laser is switched on a temperature gradient of 2-6° C occurs causing fluorophore excitation which is detected by a detector. The gradient ultimately causes the flurophhores deplete. Multiple capillaries with constant amount of fluorophores and increasing amount of ligand are scanned consecutively to gain the binding contansts.IR-Laser induced Fluorescence signal, thermophoresis, fast changes due to temperature in fluorescence intensity, and diffusion after laser is switched off are recorded to calculate different parameters.MST is not only one of the best available technology but it is also easy to handle, it has no limitation on molecular weight and has a low sample consumption rate as compared to ITC which makes it the more appropriate method used to