13C nuclear magnetic resonance studies of biological systems

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Abstract

Nuclear magnetic resonance techniques have been applied to the study of two biological systems. Specifically, ¹³C nmr spin-lattice relaxation times (T₁ s) have been applied to a model biomembrane and also to the subject of protein denaturation.

In the former case, available ¹³C enrichment techniques have been used to concentrate the isotope in a bacterial phospholipid.

Using T₁ as an index of molecular motion, it has been possible to observe the "dynamics" of the carbon skeleton of the enriched phosphatidyl ethanolamine in a mixed phosphatidyl ethanolamine - phosphatidyl serine system, over the temperature range of 30-85°C. The results obtained fit suggested motional gradients typical of the type of structure formed in media of low to high dielectricity. However, changes with temperature in the mixed lipid system may be less dramatic than have been reported for single component models. The estimated energy of relaxation processes show that a higher activation energy is associated with those nuclei in the middle of the acyl residues.

A method is described, for the first time, for the high ¹³C specific enrichment of phosphatidyl choline acyl residues. The method is based on the predatory growth of a ciliate on an E. coli strain which is designed to incorporate acetate efficiently.

Tetrahymena pyriformis which was the ciliate used, was found to metabolize the E. coli lipids resulting in a lipid distribution that is characteristic in normal growth. The enrichment levels found in the extracted lipids of Tetrahymena are interpreted in terms of the possible mechanisms of some steps in lipid metabolism.

The attractions in this area are two-fold; firstly the high enrichment (20-50%) in ¹³C of phosphatidyl choline which is usually the major lipid component of eukaryotes, and secondly the preservation of an "alternate-carbon” enrichment making subsequent study by ¹³C nmr simpler than it would have been if direct ¹³C-¹³C spin coupling existed.

In the subject of protein denaturation, ¹³C T₁ values are reported as a function of concentration of a protein denaturant in the presence and absence of a fixed protein concentration . No direct evidence is available for strong "ligand-type" protein-denaturant association. Weaker interactions cannot, however, be equally ruled out. More interestingly, Urea which is the denaturing agent in question is directly implicated in substantial associations with water via hydrogen bond formation. An increase in "lattice" disorder or "structural temperature” of the water is invoked to explain the "solubilization" of the protein. The possible role of different intermediates of denatured protein conformers is discussed.

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