Application of15N NMR Spectroscopy to Studies of the Intermolecular Interaction of Biomolecules

Nitrogen nuclei are frequently located at the interaction sites of biomolecules; for example, amide nitrogens in the peptide are the key to maintaining the peptide backbone conformation by hydrogen bonding. Histidine, tryptophan, and arginine side chains contain nitrogen atoms which are often located at the active sites of enzymes. Heterocyclic compounds like purine and pyrimidine bases are substances essential to information transfer by base pair formation of nucleic acids. Also some other co-factors and dyes, such as flavins, porphyrins and chlorophylls, are nitrogen-containing substances which regulate energy tranduction in biological systems. Lecithin and phosphatidylethanolamine are the principal components of the phospholipids from biomembranes. To detect interaction sites and to study the interaction mechanism of these biomolecules, the use of nitrogen NMR seems promising. Although more than 99% of naturally occurring nitrogen element has the 14N nuclei, a disadvantage of the use of 14N magnetic resonance has been recognized. It has a spin quantum number I = 1 and the associated quadrupole moment provides a very broad resonance signal of about 100∼1000 Hz. Thus, for detection of small changes of the chemical environment, use of 14N magnetic resonance is not adequate. The natural abundance of 15N nuclei with a spin quantum number I = 1/2 (which give a sharp resonance signal) is only 0.3% (Table 1). But recent developments in the instrumentation of NMR spectroscopy have made it possible to observe the resonance of the nuclei with low natural abundance. Fourier transform (FT) NMR can save thousands of times the accumulation time to improve the signal to noise ratio of 15N spectra [1-3]. Also superconducting magnets with wide bores have made possible the use of thick sample tubes of 25 mmΦ and observation of the 15N resonance of substances of low solubility [4]. In spite of such instrumental development, the observation of the 15N resonance is still not easy because of its low sensitivity; about of proton magnetic resonance. In the application of 15N NMR in biological systems, we often encounter quite low solubility of biomacromolecules and also sometimes need to measure the concentration dependency of 14N chemical shifts. For such experiments, enrichment of 15N nuclei in the molecules is required. Chemical syntheses starting from the simple 15N containing compounds as an 15N source and also biological syntheses by bacterial fermentation using the 15N source in culture media are employed for 15N enrichment. Enrichment at specific positions of biomolecules is useful for spectral assignments and also for analyses of the pathways of biosyntheses [5].