Protein-ligand interaction: a NMR study

Proteins perform numerous tasks within living organisms, some act as structural support for cells and tissues, others are transporters of non-soluble molecules in water and others, such as lysozyme, are involved in defensive mechanisms against bacterial attacks. The growing ease of obtaining proteins in substantial quantities and purity, by means of increasingly sophisticated bio-engineering techniques (1), may lead to their use in pharmaceutical formulations as drug delivery mechanisms or in food as preservatives (2). However, given the chemical nature of these macromolecules, they are vulnerable to oxidative attacks that would lead to their denaturing resulting in loss of activity. Moreover, some of these, such as lysozyme, once denatured, can give rise to aggregation leading to the onset of amyloidogenic diseases such as Alzheimer's disease and transmissible spongiform encephalopathies. The proteins that cause these diseases have no common characteristics in their native state, but once converted into amyloid fibrils they have many analogies (3). The factors that induce the formation of aggregates remain obscure; a hypothesis is the malfunctioning of mechanisms that govern the folding of proteins and the elimination of erroneously folded ones, or even oxidative stress phenomena caused by a malfunction of superoxide dismutase mutation. Therefore, it is vital to be able to prevent the oxidation of these enzymes in order to maintain their activity unaltered before they can be used on a large scale. The aim of this study is to investigate, through Nuclear Magnetic Resonance, the interaction between a protein with potential industrial applications (lysozyme), with a molecule with known antioxidant potential, the catechin, which has no compatibility issues with the human body being a substance naturally occurring in numerous foods of natural origin. Subsequently, in order to verify the protective effect of catechin on the protein, the effect of an oxidizing system on lysozyme was assessed both in the absence and the presence of catechin. Finally, in order to estimate the magnitude of such protection, a comparison with another known antioxidant system, the ascorbic acid (4), has been carried out. The study begins with the chemico-physical characterization of the molecules employing advanced spectroscopic techniques such as 1H-1H COSY, TOCSY, NOESY and 1H-13C HSQC and HMBC bidimensional experiments as well DOSY. These experiments aim to evaluate the spatial structure and arrangement of isolated molecules, as well as evaluate their translational mobility. The study proceeds with the evaluation of the interaction based on the variation of the ligand self-diffusion coefficient, is continued by determining the binding constant by acquiring spectra at different concentration ratios and finally identifying the interaction site on the protein. (1) Gossen M, Bujard H. Proc Natl Acad Sci 1992, 15, 89, 5547-5551. (2) Proctor VA., Cunningham FE, Fung YC. Food Sci. and Nut,1988; 26, 359-395 (3) Booth DR, Sunde M, Bellotti V, Robinson CV, Hutchinson WL Fraser PE, Hawkins PN, Dobson CN, Radford SE, Blake CCF, Pepys MB. Nature 385, 787-793 (4) Daojin L, Baoming J, Jing J. Elsevier,2008; 128, 1399-1406