Nuclear magnetic resonance studies of blood platelets

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.     

Nuclear magnetic resonance studies of amphiphile hydration.

The nuclear magnetic resonance signal of water which remains unfrozen at ?25 °C in the presence of phosphatidylcholine has been used to determine the hydration of this amphiphile. The effects of cholesterol and sodium dodecylsulfate on both the area and linewidth of this signal indicate that these molecules cause significant changes in the structure of phosphatidylcholine vesicles in solution. Studies on other amphiphiles indicate that, whereas phosphatidylethanolamine has a hydration similar to phosphatidylcholine, species with just one hydrocarbon chain such as sodium dodecylsulfate and dodecyltrimethylammonium bromide have little, if any, hydration when assayed via the nuclear magnetic resonance experiment.     

Nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome c'

Cytochrome c' from Rhodospirillum rubrum has been studied by proton magnetic resonance (NMR) at 270 MHz. The pH and temperature-dependence properties as well as proton water relaxation enhancement and bulk susceptibility measurements were examined. We conclude that the fifth ligand to the iron is histidine. The pH-dependent shift of the heme methyl resonances of the ferric protein shows pKa's at 5.8 and 8.7. The low-pH equilibrium causes only minor changes in the properties of the protein. However, the high-pH equilibrium causes large changes throughout the NMR spectra which correlate with the reported visible spectral changes. These NMR spectral changes are compared with the low-temperature EPR and M?ssbauer spectroscopic data. Analyses of the NMR data show that a second histidine, which is present in the sequence of c' from R. rubrum but is not conserved in other cytochromes c', is not a "distal" histidine. The nature of the sixth ligand and the significance of the high-pH transition are discussed.     

Nuclear magnetic resonance studies of polyisoprenols in model membranes

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locale within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C10), farnesol (C15), and solanesol (C45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled prenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C95) and undecaprenol (C55). 2H-NMR results show that the head and tail 2H-labeled sites of C55 and C95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C55 and C95 either have substantially different (faster) motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C55 and C95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.     

Nuclear magnetic resonance studies of the denaturation of ubiquitin.

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.     

Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.