Martin Raymond Jefson

Chapter 28. Applications of NMR Spectroscopy to Protein Structure Determination

Publisher Summary This chapter discusses the experimental procedures and computational methods used in the determination of the spatial conformation of protein molecules. Topics such as conformational states and protein dynamics, receptor–ligand interactions, use of NMR and site-directed mutagenesis, and future directions have also been discussed in the chapter. With the advent of high magnetic field spectrometers and two dimensional techniques, nuclear magnetic resonance spectroscopy (NMR) now compliments crystallography as a means of determining macromolecular conformations. NMR provides the opportunity to study the conformation of proteins in noncrystalline environments, most notably in aqueous solution or in micelles. The emergence of two dimensional NMR methods has greatly simplified this exercise. The NMR data can further be used to define the secondary structure of the protein. Finally, a three-dimensional structure can be determined, using constraints on inter-atomic distances and backbone torsional angles derived from the NMR data. Two-dimensional (2D) NMR methods achieve the necessary simplification, and their use has allowed the three-dimensional structure of proteins up to approximately 10 kDa in size to be solved. At present, it is possible to accurately determine the global polypeptide fold in solution of proteins up to 10 kDa in size, using a combination of NMR spectroscopy and computational methods. Two significant limitations of this approach are its lack of accuracy in the determination of localized structure and size limitation.

Chapter 11. Antibacterial Agents

Publisher Summary Research continues to search within known antibiotic classes for improved agents, look for novel classes lacking cross-resistance, and study resistance mechanisms to approach the problem of resistant bacteria and the possible consequences. This chapter discusses the important developments of the recent times related to β-lactams, quinolones, tetracyclines, and macrolides, in addition to several less-developed areas that offer potential improvements in therapeutic options. New information, pertaining to the mechanisms of quinolone activity and toxicity, has also appeared. Topoisomerase IV may be a target for the quinolones in intact bacteria, and the accumulation of quinolones is significantly increased in post-antibiotic effect (PAE) phase cells relative to the actively growing cells. The potency of quinolones against mammalian topoisomerase II is, to a large extent, dependent on the structure of the C-7 substituent. A number β-lactams, with unusual structures, has been recognized. E1101, the isopropoxycarbonyloxymethyl ester prodrug of E1100 exhibits in effect oral absorption in laboratory animals and humans and has been the subject of an extensive chemistry and structure–activity relationship (SAR) study. The increased need for new agents for combating antibiotic-resistant bacteria has rejuvenated interest in the tetracycline class. Two common mechanisms of resistance to tetracyclines exist. The first is the removal of drug from the bacterial cell by membrane-bound efflux proteins and the second is the ribosomal protection that prevents tetracyclines from interrupting polypeptide chain elongation. The well-precedented ability of macrolides to penetrate and accumulate in various host cells has led to: (1) studies of the utility of macrolides versus infections, caused by intracellular pathogens (for example, chlamydia and enteric bacteria) and (2) further studies on uptake and effect on macrophages, neutrophils, and T-lymphocytes.