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J M Beechem

Publications and source records attributed to J M Beechem.

At least 55 records · Page 3Linked to original sources
PubMed1993

Influence of 5'-nearest neighbors on the insertion kinetics of the fluorescent nucleotide analog 2-aminopurine by Klenow fragment.

The effects of nearest neighbor interactions between a nucleotide base at the primer 3'-terminus and an incoming deoxyribonucleoside triphosphate on DNA polymerase catalyzed insertion were examined. Kinetics of inserting the fluorescent nucleotide analog 2-aminopurine deoxyribonucleotide (dAPMP) and dAMP opposite a template T by 3'-->5' exonuclease-deficient mutants of Klenow fragment (KF-) were measured on primer/templates of identical sequence except for the base pair at the 3'-primer terminus. In addition to its fluorescence properties, 2-aminopurine (AP) is an attractive probe because it is misinserted opposite T by polymerases at much higher frequencies than natural nucleotides. Misinsertion frequencies for AP are on the same order of magnitude as variations in misinsertion frequencies due to changes in local DNA sequence, which makes the statistical significance of these variations easier to document. We have established that changes in the fluorescence of AP can be used to follow the insertion of dAPMP on both steady-state and pre-steady-state time scales. Rates of insertion of dAPMP measured by fluorescence and by a polyacrylamide gel assay were similar and are sensitive to the identity of the base at the 3'-primer twice as fast as insertion following a primer terminus T. The difference in rates arises primarily from differences in kcat values, which were fastest next to G and slowest next to T, while apparent Km values were similar next to each of the 4 different nearest neighbors. The gel assay was used to measure AP misinsertion efficiencies by two methods: (1) by having dAPTP and dATP directly compete for insertion opposite T in the same reaction and (2) by measuring Vmax/Km values for each substrate in separate reactions. The results from the direct competition and separate kinetics measurements are similar. The misinsertion efficiency of dAPMP relative to dAMP opposite a template T was significantly higher next to a 3'-primer terminus G (f(ins) = 0.31 +/- 0.06) than next to T (f(ins) = 0.15 +/- 0.03) for the KF- single mutant (D42A). The corresponding misinsertion efficiencies next to a 3'-primer terminus G and T were 0.20 +/- 0.02 and 0.16, respectively, for the KF- double mutant (D355A, E357A). Relative rates of insertion of dAPMP and dAMP correlate with melting temperatures calculated for nearest neighbor doublets which reflect the relative base-stacking energies. In addition to changes in insertion kinetics, polymerase-DNA dissociation rates varied with the identity of the 3'-primer terminus, differing by as much as 7-20-fold depending on the polymerase and the primer/template.

2-Aminopurine↗
PubMed1993

Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1993

alpha 2-Macroglobulin bait region integrity. Role in determining fast-form structure.

To determine whether integrity of the bait region affects the structure of the remainder of human alpha 2-macroglobulin (alpha 2M), we have determined the separation between cysteine residues in a methylamine-reacted fast-form of alpha 2M. From reduction in fluorescence intensity of covalently-bound donor fluorophore caused by proximity to an acceptor, a separation of 35 +/- 8 A was calculated, which is identical to a previously determined value for proteinase-treated fast-form alpha 2M. This indicates that although bait region cleavage is the physiological route to conformational change in alpha 2M, bait region integrity per se does not significantly affect the structure of fast-form alpha 2M.

Cysteine↗
PubMed1993

Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl.

Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated with Mal-ANS suggested that the inhibition of transport was due to a partial channel-blocking effect, and not to competition for the substrate binding site or to hindrance of carrier conformational change. In purified glucose carrier prepared from cells labeled on the exofacial sulfhydryl with Mal-ANS, a blue shift in the peak of fluorescence indicated that the fluorophore was in a relatively hydrophobic environment. Mal-ANS fluorescence in such preparations was quenched by ligands with affinity for the outward-facing carrier (ethylidene glucose, D-glucose, and maltose), but not by inhibitors considered to bind to the inward-facing carrier conformation (cytochalasin B or phenyl beta-D-glucoside). The effect of ethylidene glucose appeared to be related to an interaction with the glucose carrier, since the concentration dependence of ethylidene glucose-induced quench correlated well with the ability of the sugar analog to inhibit cytochalasin B binding to intact cells. The hydrophilic quenchers iodide and acrylamide decreased carrier-bound Mal-ANS fluorescence, resulting in downward-curving Stern-Volmer plots. Whereas ethylidene glucose enhanced iodide-induced quench, it had no effect on that of acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1993

Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: determination of the rate-limiting step.

The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2-3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2-3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphorylation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2-3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 microM and a Vmax of 0.1 nmol/min/oocyte. Co-injection of yeast hexokinase increased glucose phosphorylation by five-fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25-30-fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2-3H]glucose were analyzed by a multiple curve-fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate-limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three-fold lower extracellular concentration of 2-deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models.

Animals↗
PubMed1993

Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin.

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.

Biophysical Phenomena↗
PubMed1993

Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data.

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.

Biophysical Phenomena↗
PubMed1993

Designing matrix models for fluorescence energy transfer between moving donors and acceptors.

A recipe is given for designing theoretical models for donor-acceptor systems in which fluorescence energy transfer and motion takes place simultaneously. This recipe is based on the idea that a system exhibiting both motion and fluorescence energy transfer can be modeled by specifying a number of "states" and the rates of transitions between them. A state in this context is a set of specific coordinates and conditions that describe the system at a certain moment in time. As time goes on, the coordinates and conditions for the system change, and this evolution can be described as a series of transitions from one state to the next. The recipe is applied to a number of example systems in which the donors and/or acceptors undergo either rotational or translational motion. In each example, fluorescence intensities and anisotropies for the donor and acceptor are calculated from solutions of eigensystems. The proposed method allows for analyzing time-resolved fluorescence energy transfer data without restrictive assumptions for motional averaging regimes and the orientation factor. It is shown that the fluorescence quantities depend on the size of the motional step (i.e., on the number of states), only if fluorescence energy transfer occurs. This finding indicates that fluorescence energy transfer studies may reveal whether the dynamics of a system (e.g., a protein) is better described in terms of transitions between a relatively small number of discrete states (jumping) or a large number of dense states (diffusion).

Biophysical Phenomena↗
PubMed1991

Orientational distribution of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles as determined by global analysis of frequency domain fluorimetry data.

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.

Diffusion↗
PubMed1990

Separation and localization of the four cysteine-949 residues in human alpha 2-macroglobulin using fluorescence energy transfer.

By use of the intermediate form (I-form) [Gettins, Crews, & Cunningham (1989) Biochemistry 28, 5613-5618], alpha 2-macroglobulin can be specifically labeled with fluorescent probes in a manner that allows the determination of the topology of the four thiol ester derived Cys949 residues within this large tetrameric protease inhibitor. Freshly prepared I-form alpha 2-macroglobulin was reacted with 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonate (1,5-I-AEDANS) to produce alpha 2-macroglobulin specifically and stoichiometrically labeled with 1,5-AEDANS (donor) at the two Cys949 SH groups in the first protease interaction site. Upon subsequent reaction of this labeled species with chymotrypsin, the remaining two bait regions and thiol ester linkages were opened, generating two free SH groups on the two Cys949 residues in the second protease interaction site. These SH groups were specifically and stiochiometrically labeled with 5-(iodoacetamido)fluorescein (acceptor). Fluorescence energy transfer from donor to acceptor results in 82% loss of AEDANS fluorescence intensity. By use of an R0(2/3) value of 43.5 A, calculated from the spectral parameters of this system, an R(2/3) separation between donor and acceptor of 33.9 A was calculated. From fluorescence anisotropy measurements of both donor and acceptor attached to alpha 2-macroglobulin, upper and lower limits on the separation of 43.4 and 26.1 A, respectively, were calculated. These separations, small in the context of the alpha 2-macroglobulin tetramer, which has approximate dimensions of 190 x 90 x 90 A, severely restrict the possible locations of the four Cys949 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Analysis of binding in macromolecular complexes: a generalized numerical approach.

In this work, we introduce a generalized, global numerical methodology for analysis of binding phenomena in complex macromolecular assemblies. On the basis of a numerical algorithm (EQS) to solve systems of simultaneous free energy equations, binding profiles of simple to highly complex interacting systems can be analyzed over any concentration region without any need to generate an analytical form to describe the data. The output of the numerical algorithm is the concentration of each individual species in solution, allowing the generation of all possible binding profiles of the system (e.g., protein saturation by ligand). We present here the application of this approach to the DNA-protein subunit-ligand interactions of the trp repressor system as a typical example. From a practical point of view, the analysis program is capable of the rapid and simultaneous analysis of multiple binding profiles in terms of internally consistent sets of free energies. Given both the enormous complexity, as well as the underlying subtlety, involved in the regulation of biological function, the present generalized approach to analyzing macromolecular binding should find wide applications.

Algorithms↗
PubMed1990

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.

Ligands↗
PubMed1990

Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study.

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).

Diffusion↗
PubMed1989

A second generation global analysis program for the recovery of complex inhomogeneous fluorescence decay kinetics.

A fluorescence spectroscopy global data analysis environment is described. Within this analysis environment, multidimensional fluorescence decay data (time and frequency domains) can be analyzed in terms of a wide variety of photophysical models. A generalized compartmental analysis structure is utilized, where one can specify the functions used to link the various compartments together. All fitting parameters may be characterized by either discrete or distributed values. Applications of these new analysis programs to the examination of phase transitions in lipid/membrane systems are described.

Data Interpretation, Statistical↗
PubMed1989

Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Fluorescence energy transfer is widely used for determination of intramolecular distances in macromolecules. The time dependence of the rate of energy transfer is a function of the donor/acceptor distance distribution and fluctuations between the various conformations which may occur during the lifetime of the excited state. Previous attempts to recover both distance distributions and segmental diffusion from time-resolved experiments have been unsuccessful due to the extreme correlation between fitting parameters. A method has been developed, based on global analysis of both donor and acceptor fluorescence decay curves, which overcomes this extreme cross-correlation and allows the parameters of the equilibrium distance distributions and intramolecular diffusion constants to be recovered with high statistical significance and accuracy. Simulation studies of typical intramolecular energy transfer experiments reveal that both static and dynamic conformational distribution information can thus be obtained at a single temperature and viscosity.

Biopolymers↗
PubMed1986

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics.

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.

Hydrogen-Ion Concentration↗