Effect of urea denaturation on tryptophan fluorescence and nucleotide binding on tubulin studied by fluorescence and NMR spectroscopic methods.

Tubulin, the major protein of microtubules, has been shown to be an example of protein undergoing multistep unfolding. Local unfolding and stepwise loss of a number of characteristic functions were demonstrated. In order to understand urea induced effects on tryptophan fluorescence and nucleotide binding on tubulin, both fluorescence and NMR techniques were used. Tubulin was denatured by different urea concentrations. The present experiments were carried out at concentrations of tubulin (to approximately 10 microM) at which most of the protein will be in the dimeric state. Quenching studies in the presence of KI suggest that all the tryptophans are fairly solvent exposed. Similar studies using acrylamide as quencher, suggest unfolding of tubulin at these protein concentrations to be an apparent two state process between the native and the completely unfolded states unlike at low concentrations where a partially folded intermediate was observed. No observable effects of the nucleotide or the metal ion on tryptophan fluorescence were observed. An attempt was made using NMR to monitor the changes in the nucleotide interaction with tubulin as the protein is unfolded by urea denaturation. No significant effects were observed in the binding of the nucleotide to tubulin by urea denaturation.

Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study.

Bis(1-analino-8-naphthalenesulfonate) (bis-ANS) is a useful probe for hydrophobic areas on protein molecules and it has been proposed that it has a general affinity for the nucleotide binding site(s). There appear to be two different classes of binding sites for bis-ANS on hexokinase and these can be tentatively assigned as primary and secondary binding sites. The rate of binding of bis-ANS at the primary binding site is fast, whereas binding at secondary site(s) is slow. The slow increase in the fluorescence intensity on binding with bis-ANS is not due to conformational change in the enzyme, which may lead to the increase in the quantum yield of the bound dye. Circular dichroism measurements indicate that there is no significant change in the secondary structure on binding with this probe. In the presence of saturating amounts of glucose, the increase in fluorescence intensity due to binding at the secondary binding site(s) is significantly lowered. This indicates that glucose-induced conformational change has been sensed by this probe. From kinetic studies, it has been observed that bis-ANS is an effective competitive inhibitor of yeast hexokinase with respect to ATP. The stoichiometry of binding of this fluorescent probe is about one per subunit at the primary site both in the presence and absence of glucose, and the dissociation constant of bis-ANS is unaffected by glucose. It is possible to decrease significantly the amount of fluorescence intensity at the primary site by nucleotides. These results indicate that bis-ANS interacts at the site where nucleotide interacts. Energy transfer experiments indicate the proximity of some tryptophan(s) and bound bis-ANS molecule(s).

Nucleotide binding to tubulin-investigations by nuclear magnetic resonance spectroscopy.

In an attempt to distinguish between the interaction of GTP and ATP with tubulin dimer, high-resolution 1H- and 31P-NMR experiments have been carried out on the nucleotides in the presence of tubulin. The location of the ATP binding sites on the protein in relation to the GTP sites is still not clear. Using NMR spectroscopy, we have tried to address this question. Evidence for the existence of a site labelled as X-site and another site (labelled as L-site) for both the nucleotides on tubulin has been obtained. It is suggested that this X-site is possibly the putative E-site. In order to gain further insight into the nature of these sites, the Mg(II) at the N-site has been replaced by Mn(II) and the paramagnetic effect of Mn(II) on the linewidth of the proton resonances of tubulin-bound ATP and GTP has been studied. The results show that the L-site nucleotide is closer to the N-site metal ion compared to the X-site nucleotide. On the basis of these results, it is suggested that the L-site of ATP is distinct from the L-site of GTP while the X-site of both the nucleotides seems to be same. By using the paramagnetic effect of the metal ion, Mn(II), at the N-site on the relaxation rates of tubulin-bound ATP at L-site, distances of the protons of the base, sugar and phosphorous nuclei of the phosphorous moiety of ATP, from the N-site metal ion have been mapped. The base protons are approximately equal to 0.8-1 nm from this metal ion site. On the other hand, the phosphorous nuclei of the phosphate groups are somewhat nearer (approximately equal to 0.4-0.5 nm) from the N-site metal ion.

Interaction of Ant-ATP with tubulin: evidence for ATP competition for the GTP E-site on tubulin.

The interaction of the ribose-modified ATP analogue 3'-O-anthraniloyl adenosine 5'-triphosphate (Ant-ATP) with tubulin has been studied using steady-state fluorescence techniques. This analogue inhibits the polymerization of tubulin induced by ATP or GTP. When this analogue binds to tubulin, an increase in the fluorescence intensity of the analogue and a blue shift of about 10 nm in the emission maximum have been observed. It has been found that Ant-ATP binds to tubulin at a single binding site in the hydrophobic region with a dissociation constant of 0.5-1.0 microM. It has been possible to restore the fluorescence emission intensity of the tubulin-bound analogue to that of the free ligand with a concomitant shift in the wavelength of emission maximum to that of the free analogue by displacing the analogue with ATP or GTP. These results can be interpreted to suggest that ATP and GTP compete for the Ant-ATP binding site and that ATP binds at the GTP exchangeable site (E-site).

Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation.

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance.

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of water in cysts hydrated with D(2)O have been measured for hydrations between 1.5 and 0.1 g of D(2)O per gram of dry solids. When the relaxation rates (I/T(1), I/T(2)) of (2)H and (17)O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D(2)O (or H(2) (17)O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The (2)H and (17)O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H(2)O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T(1) as a function of hydration in the vicinity of 0.6 g H(2)O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

The nature and origin of chemical shift for intracellular water nuclei in artemia cysts.

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and (17)O nuclei.We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei ((1)H, (2)H, and (17)O) is due to significant water-macromolecular interactions.

Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase.

Our previous studies have shown that one manganous ion binds tightly to bovine hexokinase, with a Kd = 25 +/- 4 microM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (epsilon b) is 3.5 at 9 MHz and 23 degrees C [Jarori, G.K. Kasturi, S.R., and Kenkare, U.W. (1981) Arch. Biochem. Biophys. 211, 258-268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E X Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme X nucleotide X Mn. Addition of glucose 6-phosphate to enzyme X ATP X Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E X ADP X Mn X Glc-6-P appears to be formed when both the products are present. Beta, gamma-Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.

Evidence for chemically shifted water in the brine shrimp, (Artemia): possible interpretations.

We report in this paper evidence for the existence of an unusually large chemical shift for intracellular water proton in brine shrimp (Artemia). The observed chemical shift, when corrected for bulk susceptibility, is down field 0.11 ppm from the pure water signal. The possible origin of such large chemical shift for intracellular water in this system is discussed.

Factors influencing the water proton relaxation in nuclear fractions from tissues of normal and tumor-bearing animals.

Water proton spin-lattice relaxation times (T1) were measured in nuclear fractions from liver, kidney, and spleen tissues from normal and tumor-bearing animals (TBA) at two frequencies. At 9 MHz, the T1 values for TBA nuclear fractions are less than those for normal nuclear fractions, contrary to observations at the cellular level. Trace metal ion concentration and nucleic acid content estimated for these nuclear fractions suggest that the DNA-metal ion interaction might be responsible for the observed differences in the T1 of nuclear fractions at low frequencies.

Distinction between normal and leukemic bone marrow by water protons nuclear magnetic resonance relaxation times.

Pulsed nuclear magnetic resonance studies have been carried out on bone marrow of normal human subjects and patients with leukemia: chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). It was observed that the proton spin-lattice relaxation time (T1) value was discriminatory in the normal and leukemic cases with a statistical significance of (p less than 0.01). Ouabain treatment of cells did not show any perceptible change of T1 value when compared with the nontreated cells, indicating that the concomitant cation effluxes do not affect spin-lattice relaxation time. The water contents of normal, leukemic, and ouabain treated cells were in the range 60%-80%. Higher Fe levels were encountered in the normal than the leukemic samples, while levels of Zn, Cu, Mn, Co, and Ni were elevated in the leukemic samples compared with the normals. Despite the T1 differences observed, the multiparameter studies do not uniquely pinpoint factors responsible for the elevation of T1 in the malignant state.

Evaluation of muscle degeneration in inherited muscular dystrophy by nuclear magnetic resonance techniques.

Nuclear magnetic resonance (NMR) techniques were applied to study the muscular dystrophy in chicks. The water proton spin-lattice relaxation times (T1) of fast, slow, and mixed muscles and plasma were measured. The T1 values of dystrophic pectoralis major and posterior latissimus dorsi (PLD) were significantly higher than those of the normal pectoralis and PLD muscles. The present results establish a direct relationship between the differences in T1 values and the severity of muscle degeneration. Consistent with this conclusion, it was also found that the T1 values of muscles unaffected in muscular dystrophy, namely, the gastrocnemius, and anterior latissimus dorsi (ALD), were not different between the normal and dystrophic chicks. Although the affected muscles of dystrophic chicks contained higher percent water and fat than those of normal chicks, the results show that the higher T1 values in dystrophic muscles were not solely due to variations in their water content. The increase in the T1 values is principally a result of altered interaction between cellular water and macromolecules in the diseased muscles. These data also point out the potential use of NMR imaging in evaluating muscle degeneration.

Study of anisotropy in nuclear magnetic resonance relaxation times of water protons in skeletal muscle.

The anisotropy of the spin-lattice relaxation time (T1) and the spin-spin relaxation times (T2) of water protons in skeletal muscle tissue have been studied by the spin-echo technique. Both T1 and T2 have been measured for the water protons of the tibialis anterior muscle of mature male rats for theta = 0, 55, and 90 degrees, where theta is the orientation of the muscle fiber with respect to the static field. The anisotropy in T1 and T2 has been measured at temperatures of 28, -5 and -10 degrees C. No significant anisotropy was observed in the T1 of the tissue water, while an average anisotropy of approximately 5% was observed in T2 at room temperature. The average anisotropy of T2 at -5 and -10 degrees C was found to be approximately 2 and 1.3%, respectively.