NMR METHODS FOR PROTEIN BINDING STUDIES

NMR Methods for Protein Binding Studies

NMR Methods for Protein Binding Studies

Introduction

Nuclear magnetic resonance (or NMR) is a diagnostic test that provides a safe clearer view inside the human body. An NMR is different than many other diagnostic testing systems. Resonance produces magnetic images of two or three dimensions (2D or 3D) using a large magnet, radio waves and a workstation. It does not perform using x-rays. (Gardner 1997, 1389)

Over the past 20 years, advances in structural biology have profoundly changed the way in which drugs are designed and discovered. Today, instead of treating protein targets as simple black boxes, pharmaceutical scientists are now treating them as complex molecular entities. These complex molecular entities possess well-defined structures with active sites that can be rationally activated or deactivated with small molecule ligands. Innovations in structure determination methods along with rapid advances in molecular visualization tools have led to the emergence of structure-aided drug design or rational drug design as an integral part of the drug discovery and development process. Of course, the rational design of any drug depends on a precise knowledge of the 3D structure of the protein target. To date, the most prominent technique for 3D structure determination has been X-ray crystallography. No other method of structure determination can match X-ray crystallography's capacity for generating the structures of important proteins or protein complexes such as polymerases, proteosomes, viruses, and ribosome. However, not all proteins are conducive to crystallization and not all proteins behave or look the same in a crystalline state as they do in the cellular milieu. In this regard, nuclear magnetic resonance (NMR) spectroscopy has emerged as an important alternative to X-ray crystallography as it allows protein structures to be determined in conditions that are very close to the physiological state (i.e. in solution). NMR spectroscopy provides structural biologists the opportunity to measure events or processes that cannot readily be seen or quantified by X-ray crystallography, such as protein kinetics, dynamics or thermodynamics. The best known example is that of Aspirin and oral anticoagulants. Oral anticoagulants have a low therapeutic index and have effects on the developing embryo (teratogenesis). These drugs are administered in patients with cardiovascular problems or who have undergone surgery. The key thing for these patients maintain their blood fluid to prevent the formation of abnormal blood clots. All patients who use these anticoagulants are always under the close supervision of a physician, who shall calibrate the dose of anticoagulant according to the needs of each patient (this explains the dangers of these drugs). Suppose a patient under treatment with an anticoagulant present a slight headache and spontaneously take one tablet of Aspirin to alleviate it.

Acetylsalicylic acid has an affinity for plasma proteins by 98%, then goes to stand in the place of anticoagulant in the binding site (displacement). The competition between anticoagulant and aspirin then causes a sudden increase in plasma concentration of anticoagulant in a free form, with the possible risk of a hemorrhagic ...