Erik Persson

Cell water dynamics on multiple time scales

Water–biomolecule interactions have been extensively studied in dilute solutions, crystals, and rehydrated powders, but none of these model systems may capture the behavior of water in the highly organized intracellular milieu. Because of the experimental difficulty of selectively probing the structure and dynamics of water in intact cells, radically different views about the properties of cell water have proliferated. To resolve this long-standing controversy, we have measured the 2H spin relaxation rate in living bacteria cultured in D2O. The relaxation data, acquired in a wide magnetic field range (0.2 mT–12 T) and analyzed in a model-independent way, reveal water dynamics on a wide range of time scales. Contradicting the view that a substantial fraction of cell water is strongly perturbed, we find that ≈85% of cell water in Escherichia coli and in the extreme halophile Haloarcula marismortui has bulk-like dynamics. The remaining ≈15% of cell water interacts directly with biomolecular surfaces and is motionally retarded by a factor 15 ± 3 on average, corresponding to a rotational correlation time of 27 ps. This dynamic perturbation is three times larger than for small monomeric proteins in solution, a difference we attribute to secluded surface hydration sites in supramolecular assemblies. The relaxation data also show that a small fraction (≈0.1%) of cell water exchanges from buried hydration sites on the microsecond time scale, consistent with the current understanding of protein hydration in solutions and crystals.

Time scales of water dynamics at biological interfaces: peptides, proteins and cells

Water 2H and 17O spin relaxation is used to study water dynamics in the hydration layers of two small peptides, two globular proteins and in living cells of two microorganisms. The dynamical heterogeneity of hydration water is characterized by performing relaxation measurements over a wide temperature range, extending deeply into the supercooled regime, or by covering a wide frequency range. Protein hydration layers can be described by a power-law distribution of rotational correlation times with an exponent close to 2. This distribution comprises a small fraction of protein-specific hydration sites, where water rotation is strongly retarded, and a dominant fraction of generic hydration sites, where water rotation is as fast as in the hydration shells of small peptides. The generic dynamic perturbation factor is less than 2 at room temperature and exhibits a maximum near 260 K. The dynamic perturbation is induced by H-bond constraints that interfere with the cooperative mechanism that facilitates rotation in bulk water. Because these constraints are temperature-independent, hydration water does not follow the super-Arrhenius temperature dependence of bulk water. Water in living cells behaves as expected from studies of simpler model systems, the only difference being a larger fraction of secluded (strongly perturbed) hydration sites associated with the supramolecular organization in the cell. Intracellular water that is not in direct contact with biopolymers has essentially the same dynamics as bulk water. There is no significant difference in cell water dynamics between mesophilic and halophilic organisms, despite the high K+ and Na+ concentrations in the latter.

Nanosecond to Microsecond Protein Dynamics Probed by Magnetic Relaxation Dispersion of Buried Water Molecules

Large-scale protein conformational motions on nanosecond-microsecond time scales are important for many biological processes, but remain largely unexplored because of methodological limitations. NMR relaxation methods can access these time scales if protein tumbling is prevented, but the isotropy required for high-resolution solution NMR is then lost. However, if the immobilized protein molecules are randomly oriented, the water 2H and 17O spins relax as in a solution of freely tumbling protein molecules, with the crucial difference that they now sample motions on all time scales up to approximately 100 micros. In particular, the exchange rates of internal water molecules can be determined directly from the 2H or 17O magnetic relaxation dispersion (MRD) profile. This possibility opens up a new window for characterizing the motions of individual internal water molecules as well as the large-scale protein conformational fluctuations that govern the exchange rates of structural water molecules. We introduce and validate this new NMR method by presenting and analyzing an extensive set of 2H and 17O MRD data from cross-linked gels of two model proteins: bovine pancreatic trypsin inhibitor and ubiquitin. We determine residence times and order parameters of four internal water molecules in these proteins and show that they are quantitatively consistent with the information available from crystallography and solution MRD. We also show how slow motions of side-chains bearing labile hydrogens can be monitored by the same approach. Proteins of any size can be studied at physiological hydration levels with this method.

Influence of mitochondrial inhibition on global and local [Ca(2+)](I) in rat tail artery.

Inhibition of oxidative metabolism is often found to decrease contractility of systemic vascular smooth muscle, but not to reduce global [Ca2+]i. In the present study, we probe the hypothesis that it is associated with an altered pattern of intracellular Ca2+ oscillations (waves) influencing force development. In the rat tail artery, mitochondrial inhibitors (rotenone, antimycin A, and cyanide) reduced &agr;1-adrenoceptor–stimulated force by 50% to 80%, but did not reduce global [Ca2+]i. Less relaxation (about 30%) was observed after inhibition of myosin phosphatase activity with calyculin A, suggesting that part of the metabolic sensitivity involves the regulation of myosin 20-kDa light chain phosphorylation, although no decrease in phosphorylation was found in freeze-clamped tissue. Confocal imaging revealed that the mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular Ca2+ waves elicited by &agr;1 stimulation. The altered wave pattern, in association with increased basal [Ca2+]i, accounted for the unchanged global [Ca2+]i. Inhibition of glycolytic ATP production by arsenate caused similar effects on Ca2+ waves and global [Ca2+]i, developing gradually in parallel with decreased contractility. Inhibition of wave activity by the InsP3 receptor antagonist 2-APB correlated closely with relaxation. Furthermore, abolition of waves with thapsigargin in the presence of verapamil reduced force by about 50%, despite unaltered global [Ca2+]i, suggesting that contraction may at least partly depend on Ca2+ wave activity. This study therefore indicates that mitochondrial inhibition influences Ca2+ wave activity, possibly due to a close spatial relationship of mitochondria and the sarcoplasmic reticulum and that this contributes to metabolic vascular relaxation.

Study of water dynamics and distances in paramagnetic solids by variable-temperature two-dimensional H2 NMR spectroscopy

A recently proposed two-dimensional (2)H NMR experiment is used to measure the (2)H (spin I=1) quadrupolar and paramagnetic shift anisotropy interactions in powdered CuCl(2).2D(2)O as a function of temperature. The principal components of the quadrupolar and paramagnetic shift anisotropy tensors and the Euler angles describing the orientations of the tensors in the molecular frame are determined at each temperature. For this purpose an analytical approach is introduced to extract desired parameters from motionally averaged two-dimensional line shapes where the averaging is introduced by rapid 180 degrees flips around C(2) axes of D(2)O molecules. This approach can be readily applied to study various materials containing water of crystallization. It is also clearly shown that the rapid continuous rotation of D(2)O molecules around their C(2) axes is not taking place in the studied solid in the range of temperatures between 209 and 344 K. Once the paramagnetic shift anisotropy of a deuterium atom is measured accurately it is used to estimate the distance between deuterium and the nearest copper atom bearing an unpaired electron. Excellent agreement is found between structural parameters obtained in this study and those provided by neutron and x-ray diffraction, showing that the paramagnetic shift anisotropy is a sensitive probe of distances in paramagnetic solids.

Citizens of Mars Ltd.

When the time comes to decide how to govern an extraterrestrial settlement there will be many alternatives to chose from. We will have the opportunity to try new and so far untested theories, but there are also some old forms of government that might be tempting to try again. We might for instance let the company whose activities on the world are the reason for the establishment govern the settlement. This has been tried before on our own planet both because it was seen as convenient and as an incentive for colonisation. In this chapter I will ask what this solution would mean for the civil liberty of the settlers. To answer the question I will look at some historical analogues and have a philosophical discussion. The conclusion is that a settlement governed by a body whose sole reason for existence is to make money for the owners, that is led by a board that answers only to the owners and not to the people, that functions as both government and sole employer, and that has the unlimited power over the life support systems necessary for the survival of the settlers will not be a good basis for civil liberties.