K+ RIBOSOME INTERACTIONS DETERMINE THE LARGE ENHANCEMENTS OF K-39 NMR TRANSVERSE RELAXATION RATES IN THE CYTOPLASM OF ESCHERICHIA-COLI K-12

As a probe of physical chemical properties of the intracellular environment, we measured K-39 NMR transverse relaxation rates in concentrated cell slurries of Escherichia coli K-12 grown in minimal medium over a range of osmolarities (from 0.1 to 1.0 OsM) and after plasmolysis. The K-39 transverse relaxation at a resonance frequency of similar to 18.67 MHz is biexponential under all conditions, and 100% of the expected signal intensity is detected. Both components of the K-39 NMR transverse relaxation are very fast, and the difference between the fast and slow relaxation rates is very large compared to previous measurements on Na-23 and K-39 in protein and nucleic acid solutions in vitro. The K-39 transverse relaxation rates decrease as the osmolarity of the growth media increases but increase dramatically when cells grown in 0.1 OsM media are plasmolyzed at 1.0 OsM. The homogeneous nature and the 100% visibility of the K-39 signal indicate the existence of fast exchange among the multiple, magnetically distinguishable populations of K-39 which probably exist in the cytoplasm. The absence of static quadrupolar splitting of the cytoplasmic K-39 signal (as indicated by a single peak in the spectrum) indicates that the cytoplasm, as probed by K-39 NMR, behaves Like a concentrated but isotropic nucleic acid solution rather than an anisotropic nucleic acid liquid crystal. To understand the origins of the striking NMR relaxation behavior of K-39 in viable cells, we have investigated NMR transverse relaxation rates of K-39 (and also Na-23 and Cl-35) in E. coli 50S and 70S ribosome solutions in vitro. At concentrations of ions and of ribosomes that to the extent possible mimic those of the cytoplasm of E. coli, we find that K-39, Na-23, and Cl-35 transverse relaxation rates all exhibit biexponential behavior, and K-39 and Na-23 exhibit the large magnitudes and the large difference between the slow and the fast relaxation rates observed in viable cells. These polyanionic ribosome solutions are the only in vitro model system discovered to date that exhibits K-39 transverse relaxation rates comparable to those in viable cells. We conclude that K+-ribosome interactions are the dominant source of the NMR properties of K+ in E. coli. We propose that the relatively small dependence of in vivo K-39 transverse relaxation rates in the cytoplasm of E. coli on cytoplasmic K+ concentration (increased by increasing the osmolarity of the growth medium) results from near-compensation between two significant contributions: (1) the ratio of K+-to-nucleic acid phosphate increases, tending to decrease the K-39 transverse relaxation rates, and (2) the concentration of ribosomal particles increases because the volume of cytoplasmic water decreases, tending to increase the K-39 transverse relaxation rate. We propose that the dramatic increase observed in the K-39 transverse relaxation rates in plasmolyzed cells results from the increase in cytoplasmic ribosome concentration at constant K+: phosphate ratio and the large decrease in cytoplasmic water upon plasmolysis.