The role of sophisticated instruments in biological research: Examples from nuclear magnetic resonance

The development of NMR is described to illustrate the importance of new methodologies to solve biological problems.     

Organo-bridged dicobaloximes: Synthesis, structure and nuclear magnetic resonance study

This short review describes the synthesis and structural characteristics of methylene, xylylene and biphenyl-bridged dicobaloximes. The bridged dicobaloximes are easily synthesized by oxidative alkylation method and the yield of dicobaloxime depends on many factors like reaction time, solvent, dihalide:Co(I) ratio and also on the nature of dihalide used. The NMR and structural studies have been described. The cobalt-bound CH₂ and dioxime protons in xylylene-bridged dicobaloximes become nonequivalent at sub-zero temperature and this is caused by the restricted rotation of the Co-C bond. However, the nonequivalence in biphenyl-bridged dicobaloximes is caused by atropisomerism and/or restricted rotation of C-Ph/Co-C bond. Two cobaloxime units of biphenyl-bridged dicobaloximes are symmetrical, and can be looked at as two independent benzyl cobaloximes and there is no direct or indirect interaction between them. In the xylylene-bridged dicobaloxime, one cobaloxime unit affects the structural parameters of the other unit through axially bridged benzyl group, which is sandwiched between two dioxime wings.     

Ribosome-chloramphenicol interactions: a nuclear magnetic resonance study

Proton magnetic resonance line broadening of chloramphenicol resonances has been employed to study the binding of this inhibitor of protein synthesis to the Escherichia coli 70 S ribosome. Temperature dependence measurements of the resonance line widths show that chloramphenicol is in fast exchange with the ribosome. Differential broadening of the various drug resonances suggests that it binds in its receptor site in essentially the same conformation that exists free in solution. Thus, even though the drug possesses a fair degree of structural flexibility, this is not necessary for its interaction with the ribosome. The recognition is most likely of the classic lock and key type, with the ribosomal site being essentially an open gate for the fitting of the drug. From the proton line-width measurements and ¹⁹F spectra of a derivative, it has been possible to propose a model for the geometry of chloramphenicol when it resides on the ribosome which is consistent with established structure-activity relationships for the drug.     

Conformational analysis by nuclear magnetic resonance: insulin

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).     

Applications of nuclear magnetic resonance in parasitology

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical framework with emphasis on magnetic nuclei of 1/2 spin, including 1H, 13C, and 31P. Biological applications of NMR spectroscopy and magnetic resonance imaging (MRI) are outlined briefly. NMR spectroscopic studies on parasitic protozoa, cestodes, nematodes, trematodes, and hymenopterous insect parasites are reviewed. NMR and MRI investigations on the pathophysiology of the host are also discussed, and the potential future of NMR applications in parasitology outlined.     

Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase.

The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.     

Isolation,purification and nuclear magnetic resonance spectra of pulvilloric acid

A strain ofPenicillium pulvillorum which produces pulvilloric acid has been identified. Pulvilloric acid has been isolated and purified and modern techniques have been used to assign its¹³C and¹H nuclear magnetic resonance (NMR) spectra.     

Scaling in biological nuclear magnetic resonance spectral distributions

A statistical analysis of the distribution of the eigenvalues of the chemical shift interaction as detected by nuclear magnetic resonance (NMR) spectroscopy in large biological systems is presented in the light of random matrix theory. A power law dependence is experimentally observed for the distribution of the number of eigenvalues, N, of the shielding hamiltonian with epsilon i less than or equal to E as a function of the energy E. From this cumulative distribution of energy levels, N(E), we also obtain a density of states rho(E). The exponent of the energy variation of N(E) and rho(E) are correlated with the dimensionality of the molecular system. A crossover in the values of the exponents is found in passing from low to higher energy in the spectra. Our method classifies and reduces the chemical shift data base of proteins and also demonstrates a degree of regularity in seemingly irregular spectral patterns.     

Pulsed nuclear magnetic resonance studies in sickled erythrocytes

Pulsed proton magnetic resonance studies were made to determine T₁ (spin-lattice) and T₂ (spin-spin) relaxation times for normal and sickled blood cells in the oxygenated and deoxygenated states as a function of temperature between 0°C and 37°C. It was found that the sickling phenomenon affects T₂ values but not T₁ values in these cells. These results are ascribed to a proton-exchange mechanism. The functional relationship found between T₂ and temperature in the sickled cells is similar to that between the formation of hydrophobic bonds and temperature proposed by Nalbandian and Scheraga.     

Surface plasmon resonance and nuclear magnetic resonance studies of ABAD-Abeta interaction

Abeta binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Abeta is likely a direct link between Abeta and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Abeta interaction. A van't Hoff analysis revealed that the ABAD-Abeta association is driven by a favorable entropic change (DeltaS = 300 +/- 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (DeltaH = 49 +/- 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Abeta interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Abeta. SPR confirmed the conformational changes in the ABAD-Abeta complex after Abeta binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Abeta complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Abeta on the ABAD-NAD interaction. Conversely, NAD inhibits the Abeta-ABAD interaction. Binding of Abeta and binding of NAD to ABAD are likely mutually exclusive. Thus, Abeta binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Abeta toxicity in Alzheimer's disease.     

Current trends and future directions in flower development research

Flowers, the reproductive structures of the approximately 400 000 extant species of flowering plants, exist in a tremendous range of forms and sizes, mainly due to developmental differences involving the number, arrangement, size and form of the floral organs of which they consist. However, this tremendous diversity is underpinned by a surprisingly robust basic floral structure in which a central group of carpels forms on an axis of determinate growth, almost invariably surrounded by two successive zones containing stamens and perianth organs, respectively. Over the last 25 years, remarkable progress has been achieved in describing the molecular mechanisms that control almost all aspects of flower development, from the phase change that initiates flowering to the final production of fruits and seeds. However, this work has been performed almost exclusively in a small number of eudicot model species, chief among which is Arabidopsis thaliana. Studies of flower development must now be extended to a much wider phylogenetic range of flowering plants and, indeed, to their closest living relatives, the gymnosperms. Studies of further, more wide-ranging models should provide insights that, for various reasons, cannot be obtained by studying the major existing models alone. The use of further models should also help to explain how the first flowering plants evolved from an unknown, although presumably gymnosperm-like ancestor, and rapidly diversified to become the largest major plant group and to dominate the terrestrial flora. The benefits for society of a thorough understanding of flower development are self-evident, as human life depends to a large extent on flowering plants and on the fruits and seeds they produce. In this preface to the Special Issue, we introduce eleven articles on flower development, representing work in both established and further models, including gymnosperms. We also present some of our own views on current trends and future directions of the flower development field.