Question

a. Distinguish between the commonly used methods for protein structure determination and describe the advantages and disadvantages of each.

b. Explain why immunoassays are especially useful for detecting and quantifying small amounts of a substance in a complex mixture.

c. The octapeptide AVGWRVKS was digested with trypsin. Which method would be most appropriate for separating the resulting peptides: ion exchange or gel filtration chromatography? Explain. Suppose the octapeptide was digested with chymotrypsin instead. What would be the optimal separation technique then? Explain.

Answer 1

a)Several methods are currently used to determine the structure of a protein, including X-ray crystallography, NMR spectroscopy, and electron microscopy. Each method has advantages and disadvantages. In each of these methods, the scientist uses many pieces of information to create the final atomic model.

X-ray Crystallography For this method, the protein is purified and crystallized, then subjected to an intense beam of X-rays.

NMR SpectroscopyThe protein is purified, placed in a strong magnetic field, and then probed with radio waves..

3D Electron Microscopy Electron microscopy, frequently referred to as 3DEM, is also used to determine 3D structures of large macromolecular assemblies. A beam of electrons and a system of electron lenses is used to image the biomolecule directly.

Each method has advantages and disadvantages. In each of these methods, the scientist uses many pieces of information to create the final atomic model. Primarily, the scientist has some kind of experimental data about the structure of the molecule. For X-ray crystallography, this is the X-ray diffraction pattern. For NMR spectroscopy, it is information on the local conformation and distance between atoms that are close to one another. In electron microscopy, it is an image of the overall shape of the molecule.

b) Immunoassays are based on the principles that specific antigens will stimulate very specific (unique) immune responses and that the proteins produced by the immune response, called antibodies, can be used to signal the presence of a target compound in a sample There are two types of immunoassays: sandwich and competitive. By adding known amounts of a specific sample to a detection system and measuring the amount of signal produced, a standard curve can be produced.