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Biomedical subjects

D M Jameson

Publications and source records attributed to D M Jameson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli.

Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

Anisotropy↗
PubMed1996

Mutations in a specific human serum albumin thyroxine binding site define the structural basis of familial dysalbuminemic hyperthyroxinemia.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.

Binding Sites↗
PubMed1996

Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.

The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

Binding Sites↗
PubMed1996

Synthesis and spectral characterization of sulfhydryl-reactive fluorescent probes.

We synthesized two sulfhydryl-reactive fluorescent probes, Br-ANT (2-amino-benzoic acid, 2-(bromoacetyl)hydrazide) and Br-MANT (N-[2-[(bromoacetyl)amino]ethyl]-2-(methylamino)benzamide). Br-ANT and Br-MANT contain an anthraniloyl and N-methyl anthraniloyl group, respectively, linked to the sulfhydryl-reactive bromoacetyl moiety. The cysteine adducts have absorption maxima at 323 and 326 nm, with molar extinction coefficients of 2100 and 2900 M-1 cm-1, for Br-ANT and Br-MANT, respectively, making these probes excellent acceptors for tryptophan. The absorption spectra and quantum yields were constant at pH levels useful for protein studies (pH 5-8). Quantum yields of Br-ANT and Br-MANT were 0.16 and 0.42, emission maxima were 432 and 440 nm, and fluorescence lifetimes were 1.3 and 7.8 ns, respectively. The emission of Br-ANT-Cys and Br-MANT-Cys shifted to shorter wavelengths with decreasing solvent polarity. Polarization values were maximal between 330 and 375 nm. Both probes reacted selectively and stoichiometrically with the single cysteine residue of a model protein. The labeled protein exhibited relatively long lifetimes (9-10 ns), suggesting that these probes will be generally useful for rotational studies.

Benzamides↗
PubMed1996

Spectral properties of environmentally sensitive probes associated with horseradish peroxidase.

The environmentally sensitive fluorescent probes 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) and 2'-(N,N-dimethylamino)-6-naphthoyl-4-trans-cyclohexanioc acid (DANCA) form complexes with the heme binding site of apohorseradish peroxidase. The dissociation constants of the PRODAN and DANCA complexes were determined from anisotropy titration data to be approximately 8.7 x 10(-5) and 3.3 x 10(-4) M, respectively. From comparison of the steady state fluorescence spectra of PRODAN and DANCA in solvents of varying dielectric constants, and in the apohorseradish peroxidase complex, we conclude that the heme binding site of horseradish peroxidase is relatively polar. The lifetimes of PRODAN and DANCA in organic solvents of varying polarities can be fit to single exponential decays. However, the lifetimes of PRODAN and DANCA associated with apohorseradish peroxidase, determined using a background subtraction method to correct for the non-negligible fluorescence of unbound probe, fit best to a distribution of lifetime values. We attribute these lifetime distributions to microenvironmental heterogeneity which is also consistent with the observed dependence of the emission maxima of PRODAN-apohorseradish peroxidase upon the excitation wavelength. In neither the PRODAN nor the DANCA case was evidence found in the time-resolved data for relaxation of the protein matrix around the excited state probe dipole.

2-Naphthylamine↗
PubMed1996

Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.

In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.

Animals↗
PubMed1996

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.

Acridine Orange↗
PubMed1995

Polymerization of an Escherichia coli elongation factor Tu.

Elongation factor Tu (EF-Tu) from Escherichia coli is shown here to polymerize under conditions of low ionic strength and slightly acidic pH. Several factors, such as decreasing pH, decreasing ionic strength, increasing temperature, and increasing protein concentration (up to 2.5 microM) enhance the rate of polymerization. Both EF-Tu.GTP and EF-Tu.GDP polymerized equally well under the conditions studied. A lag time was observed between the lowering of the pH and the onset of measurable polymerization, which was not overcome by addition of preformed polymer "seeds." Finally, addition of unpolymerized EF-Tu.GDP to a solution of polymerized EF-Tu.GDP appears to lead to formation of new polymers instead of addition to preexisting ones, which may suggest a size limit for polymers of EF-Tu.GDP.

Escherichia coli↗
PubMed1995

Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.

The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Transfer↗
PubMed1995

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.

Amino Acid Sequence↗
PubMed1995

Oxygen penetration and diffusion into myoglobin revealed by quenching of zincprotoporphyrin IX fluorescence.

Oxygen quenching experiments were carried out on zincprotoporphyrin IX reconstituted myoglobin (MbFe-->Zn) at different temperatures and two solvent viscosities. The data were fit to a dynamic model for quenching of fluorophores in protein interiors previously presented (Biophysical J., 45 (1984) 789-794). The parameters associated with the oxygen entry rate (k+), exit rate (k-), and migration rate (chi) in the protein were obtained at six temperatures and two viscosities (1 and 8 cp), along with the activation enthalpies associated with the above rates (k+ and k-). The partition coefficient (alpha) was calculated at each temperature along with the free energy, delta G0, associated with this partition. The rate parameters (k+, k-, chi) and the partition coefficient (alpha) have also been determined for the sample in 40% sucrose (8 cp), to evaluate the effect of bulk solvent viscosities on these values. The steady-state Stern-Volmer quenching plot was calculated using the rate parameters obtained from the analysis (of the dynamic model). Comparison of the Stern-Volmer points obtained using the dynamic model and those obtained experimentally showed excellent agreement.

Chemical Phenomena↗
PubMed1994

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.

Acrylamides↗
PubMed1994

Hydrodynamics of horseradish peroxidase revealed by global analysis of multiple fluorescence probes.

Previous fluorescence studies of horseradish peroxidase conjugated with protoporphyrin IX suggested that the protein behaved hydrodynamically as a prolate ellipsoid of axial ratio 3 to 1. The present study, designed to further investigate the hydrodynamics of this protein, exploits a series of probes, noncovalently bound to the heme binding site of apo-horseradish peroxidase, having different orientations of the excitation and emission transition dipoles with respect to the protein's rotational axes. The probes utilized included protoporphyrin IX and the naphthalene probes 1-anilino-8-naphthalene sulfonate, 2-p-toluidinyl-6-naphthalene sulfonate, and 4,4'-bis(1-anilino-8-naphthalene sulfonate). Time-resolved data were obtained using multifrequency phase fluorometry. The global analysis approach to the determination of molecular shape using multiple probes was evaluated by utilizing all data sets while maintaining a constant molecular shape for the protein. The results indicated that, in such analyses, probes exhibiting a single exponential decay and limited local motion have the major weight in the evaluation of the axial ratio. Probes that show complex decay patterns and local motions, such as the naphthalene derivatives, give rise to significant uncertainties in such global treatments. By explicitly accounting for the effect of such local motion, however, the shape of the protein can be reliably recovered.

Binding Sites↗
PubMed1994

Safety and efficacy of omega-3 fatty acids in the nutrition of very low birth weight infants: soy oil and marine oil supplementation of formula.

Because formula-fed preterm infants may be at risk of omega 3 essential fatty acid deficiency, we tested experimental formulas supplemented with soy oil to provide alpha-linolenic acid or marine oil to provide preformed omega 3 long-chain polyunsaturated fatty acids at a level comparable to that of human milk. This report addresses the effect of feeding formula supplemented with soy oil or with soy and marine oils on growth, clinical tolerance, coagulation test results, changes in erythrocyte membrane fluidity, and plasma concentrations of vitamins A and E in very low birth weight infants from 30 to 57 weeks of postconceptional age. "Healthy" preterm infants were maternally selected to receive human milk or selected at random to receive commercial ready-to-feed liquid formula, which provided limited omega 3 fatty acid, or experimental formulas supplemented with soy oil or soy and marine oils. Results of this study indicate that formula enriched with soy oil or soy and marine oils containing preformed omega 3 long-chain polyunsaturated fatty acids does not induce abnormalities in growth, clotting function, erythrocyte membrane fluidity, or vitamin A or E levels in healthy very low birth weight preterm infants. Additional studies to evaluate safety in a representative preterm population are required.

Analysis of Variance↗
PubMed1993

Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.

The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5 degrees C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30 degrees C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12]p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5 degrees C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21,000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1993

Fluorescence investigations of albumin from patients with familial dysalbuminemic hyperthyroxinemia.

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant syndrome in which clinically euthyroid patients have elevated total thyroxine levels. These high serum thyroxine levels are traceable to altered binding of thyroxine to the patient's albumin. Albumin from FDH patients and normal volunteers have been purified. Reverse-phase and ion-exchange high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the FDH-human serum albumin (HSA) samples show a single band that comigrates with normal HSA. In both protein solutions the intrinsic fluorescence, upon 280 nm excitation, is predominantly due to the single tryptophan residue. The quantum yield of this intrinsic fluorescence in the FDH-HSA solutions is, however, reduced relative to that of HSA. Furthermore, the "average" lifetime value of the tryptophan emission in the FDH-HSA sample is less than that of normal HSA, consistent with its reduced quantum yield. The binding of thyroxine to both albumins effectively quenches the tryptophan emission probably via a nonradiative energy transfer mechanism. Time-resolved data suggest that the albumin from the dysalbuminemic patients is actually an approximately equimolar mixture of normal HSA and FDH-HSA indicative of heterologous expression. Quenching of the intrinsic HSA and FDH-HSA fluorescence by serial additions of thyroxine showed enhanced quenching of FDH-HSA relative to HSA at any T4 to albumin mole ratio, therefore supporting earlier reports of increased thyroxine affinity to FDH-HSA.

Genetic Variation↗
PubMed1992

Effect of docosahexaenoic acid on membrane fluidity and function in intact cultured Y-79 retinoblastoma cells.

Considerable metabolic energy is expended in ensuring that membranes possess a characteristic fatty acid composition. The nature of the specific requirement of the retina for high levels of docosahexaenoic acid (DHA) is as yet undefined. Previous work has speculated that DHA is required to maintain the fluid nature and permeability necessary for optimal retinal function. Cultured Y-79 retinoblastoma cells were grown in serum-containing media with and without supplemental DHA. Resultant changes in membrane fluidity were assessed using fluorescent probes. No differences were observed in rotational probe mobility as assessed by fluorescence polarization despite a fourfold increase in cellular DHA content. Lateral probe mobility as assessed by pyrene eximer formation was significantly enhanced in DHA-supplemented cells. Both the DHA content and total fatty acid unsaturation index in retinoblastoma cells were directly correlated with membrane fluidity as reported by eximer formation (Pearson's rho = 0.96 and 0.92, respectively). DHA supplementation also resulted in a significant increase in cellular choline uptake. We speculate that the effect of DHA content on retinal function may be mediated by changes in membrane fluidity and associated enzyme and transport activities.

Cell Line↗