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M R Eftink

Publications and source records attributed to M R Eftink.

At least 37 records · Page 2Linked to original sources
PubMed1994

The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate.

The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C. At pH 7 and above, the unfolding transition, as monitored by changes in the fluorescence intensity at 360 nm, shows a single transition. At low pH, the transition again appears to be a single transition. In the range of 3.5-6.0, the transition is biphasic, indicating the existence of a folding intermediate. The transitions have also been studied using circular dichroism and size exclusion chromatography. The data were fitted by a model in which the dimeric protein first unfolds to form structured monomers, followed by the unfolding of the monomers. From fits with this "folded monomers" model, the free energy change for the dimer<-->monomer dissociation becomes less positive as pH is decreased; the free energy change for the unfolding of the monomers is essentially independent of pH. An alternate model is one in which the dimer first undergoes a transition to a partially unfolded dimeric state, with this intermediate then denaturing to unfolded monomers. Both models give adequate fits to the data obtained at a single protein concentration. From a study of the concentration dependence of the urea-induced unfolding at pH 5, the "folded monomers" model is found to be more consistent with the data. Size exclusion chromatography data support the description of the intermediate state, which is the most populated state at low pH in the absence of urea, as being a relatively compact monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoproteins↗
PubMed1994

Thermodynamic studies of the interaction of trp aporepressor with tryptophan analogs.

The association of L-tryptophan and some of its analogs, including three conformationally restricted analogs, with trp aporepressor (apo trpR) was studied by isothermal titration microcalorimetry. Contributions of the functional groups of a ligand to the free energy change, delta G degrees', and enthalpy change, delta H degree', of the interaction were evaluated on a molecular basis. Analogs without the alpha-amino group (i.e. desamino analogs) bind with a slightly higher affinity to the protein. On the other hand, descarboxy analogs show weaker binding to the apo trpR. In addition, it is found that there exists enthalpy-entropy compensation for the association of the congener series of ligands with the protein. The entropy change, delta S degree', appears to play a more important role in the binding of the conformationally restricted analogs than in the binding of L-tryptophan and the unlocked ligands.

Apoproteins↗
PubMed1994

The use of fluorescence methods to monitor unfolding transitions in proteins.

This article discusses several strategies for the use steady-state and time-resolved fluorescence methods to monitor unfolding transitions in proteins. The assumptions and limitations of several methods are discussed. Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species. Several examples are given, involving the unfolding of Staphylococcal aureus nuclease A, in which thermodynamic information is obtained for the temperature and denaturant induced transitions in this protein.

Biophysical Phenomena↗
PubMed1994

A multidimensional spectrophotometer for monitoring thermal unfolding transitions of macromolecules.

We describe a multidimensional spectrometer that is capable of (nearly) simultaneous measurement of circular dichroism, steady-state fluorescence, and absorbance values on the same sample in a standard 1 x 1 cm cuvette. With a computer controlled thermoelectric cell holder, this instrument is capable of measuring the above types of spectral data at various wavelengths as a function of temperature. We have developed software to control the various acquisition functions and to convert the data files to a format appropriate for use with the nonlinear least squares program, NONLIN (Johnson and Frasier, 1985). We have tested various features of this instrument and we have applied this instrument and data analysis procedure to study the thermal unfolding of ribonuclease A, under conditions were the unfolding of this protein involves an intermediate state.

Biophysical Phenomena↗
PubMed1993

Luminescence studies with trp repressor and its single-tryptophan mutants.

Time-resolved and steady-state fluorescence, low-temperature phosphorescence, and optically detected magnetic resonance (ODMR) measurements have been made to resolve the luminescence contributions of the two intrinsic tryptophan residues in the subunits of trp aporepressor from Escherichia coli. Assignments of spectral information have been confirmed by use of the single-tryptophan mutants W19F and W99F. Solute fluorescence quenching studies show that both Trp19 and Trp99 are exposed to acrylamide and iodide, with Trp99 being the more exposed. Time-resolved and steady-state fluorescence measurements show Trp19 to have a bluer emission, a longer mean fluorescence decay time, a higher quantum yield, and essentially no independent rotational motion with respect to the protein. Trp99 is found to have a redder emission, a shorter mean fluorescence decay time, a lower quantum yield, and a significant degree of rotational freedom. Phosphorescence studies show a clear resolution of 0-0 vibronic transitions for each type of residue, with maxima at 407 and 415 nm that are assigned to Trp19 and Trp99, respectively. ODMR measurements show the zero-field splitting parameters to be quite characteristically different for each tryptophan residue. The existence of resonance energy transfer from Trp19 to Trp99, in the wild-type protein, is indicated by three types of data: comparison of the long-lived decay time (attributed to Trp19) in the absence (W99F) and presence (wild type) of the acceptor Trp99, comparison of the fluorescence quantum yield of the wild-type and mutant proteins, and deviations from the expected phosphorescence intensities for Trp19 and Trp99 in the absence of energy transfer.

Apoproteins↗
PubMed1993

Interaction of indoleacrylic acid with Trp aporepressor from Escherichia coli.

Trans-beta-Indoleacrylic acid (IAA) binds with moderately high affinity to the dimeric protein, trp aporepressor from Escherichia coli. IAA is itself nonfluorescent, and, upon binding, IAA quenches approximately 95% of the intrinsic tryptophan fluorescence of the protein. Since there is an overlap between the absorbance of IAA and the emission of tryptophan, this quenching appears to be due to resonance energy transfer. To adequately fit binding isotherms, it was necessary to use a model in which the binding of IAA to one subunit is able to quench fluorescence from both subunits of the protein. This model also assumes that binding to the two subunits occurs independently. Binding data were obtained as a function of the concentration of trp aporepressor, over the range of 1 x 10(-8) to 5 x 10(-5) M, in order to test the possibility that protein subunit association or dissociation occurs. When analyzed in terms of the above model, no significant protein concentration dependence is seen for the IAA association constant, indicating that either (i) the protein does not undergo changes in oligomerization over this concentration range or that (ii) different states of aggregation bind IAA with similar affinity. The affinity of IAA was found to increase at higher ionic strength and at lower pH. Also, evidence is presented for a photoinduced change in the configuration of IAA.

Bacterial Proteins↗
PubMed1993

Local anesthetic-phospholipid interactions. Effects of ionic strength, temperature, and phospholipid mixtures on the binding of dibucaine to phospholipids.

The nature of the interaction of amphipathic drugs, such as dibucaine, with phospholipid bilayer membranes was investigated using equilibrium dialysis. Profiles for the binding of cationic dibucaine to unilamellar vesicles were obtained at different temperature and ionic strengths, and for mixtures of neutral phospholipid dimyristylphosphatidylcholine (DMPC) with negatively charged dimyristylphosphatidylglycerol (DMPG). The degree of binding of the cationic drug at pH 5 was found to be higher at temperatures above the Tm of DMPC (24 degrees C) than below Tm. Also enhanced drug binding was found to occur as the concentration of monovalent salt was increased (0.01-0.85 M) and as the percentage of DMPG was increased. Using the Stern and Guoy-Chapman model, which takes into consideration electrostatic effects, we were able to simultaneously fit all our binding data with a minimum of fitting parameters. These parameters (for data at 45 degrees C) are an association constant, K, of 330 M-1, a maximum possible number of drug molecules absorbed per unit surface of vesicle, sigma m+, of 1.70 x 10(-2) A2, and a surface area per bound drug, gamma D, of 48 A2. The data were fitted equally well by an alternate model in which binding of the drug is described as a partitioning equilibrium, with factors included for electrostatic effects and surface expansion caused by drug intercalation between the fatty acid chains.

Data Interpretation, Statistical↗
PubMed1993

Fluorescence studies of a local anesthetic-phospholipid interaction.

Steady-state and time-resolved fluorescence data are reported for the local anesthetic dibucaine in the absence and presence of phospholipid vesicles. These vesicles were comprised of dimyristylphosphatidyl choline and approximately 10% dimyristylphosphatidyl glycerol. Solute quenching studies show the bound drug to be protected from collision with iodide ion. The fluorescence lifetime of dibucaine is not significantly changed upon binding to vesicles. The fluorescence anisotropy of dibucaine increases upon association with the vesicles. Anisotropy decay measurements show that the rotational correlation time, phi, of bound dibucaine is increased about one hundred fold over that for free dibucaine. This indicates that the rotational motion of bound dibucaine is slowed by its interaction with the phospholipids. However, we find no evidence that the rotational motion of bound dibucaine is anisotropic.

Dibucaine↗
PubMed1992

Fluorescence studies with human epidermal growth factor.

Steady-state and time-resolved fluorescence studies have been performed with human epidermal growth factor, a small globular protein having two adjacent tryptophan residues near its C-terminus. Based on the relatively red fluorescence and accessibility to solute quenchers, the two tryptophan residues are found to be exposed to solvent. Anisotropy decay measurements show the dominant depolarizing process to have a sub-nanosecond rotational correlation time indicating the existence of rapid segmental motion of the fluorescing tryptophan residues. From an analysis of the low-temperature excitation anisotropy spectrum of the protein (and in comparison with that of tryptophan, the peptide melittin, and the dipeptide trp-trp), it is concluded that homo-energy transfer and/or exciton interaction occurs between the adjacent tryptophan residues. A thermal transition in the structure of the protein, which is observed by circular dichroism measurements, is not sensed by the steady-state fluorescence of the protein. This result, in conjunction with the anisotropy decay results, indicates that the two tryptophan residues are in a highly flexible C-terminus segment, which is not an integral part of the three-dimensional structure of the protein. Fluorescence measurements with three site-directed mutants also show very little variation.

Amino Acid Sequence↗
PubMed1991

Effects of temperature on the fluorescence intensity and anisotropy decays of staphylococcal nuclease and the less stable nuclease-conA-SG28 mutant.

Frequency-domain fluorescence spectroscopy was used to investigate the effects of temperature on the intensity and anisotropy decays of the single tryptophan residues of Staphylococcal nuclease A and its nuclease-conA-SG28 mutant. This mutant has the beta-turn forming hexapeptide, Ser-Gly-Asn-Gly-Ser-Pro, substituted for the pentapeptide Tyr-Lys-Gly-Gln-Pro at positions 27-31. The intensity decays were analyzed in terms of a sum of exponentials and with Lorentzian distributions of decay times. The anisotropy decays were analyzed in terms of a sum of exponentials. Both the intensity and anisotropy decay parameters strongly depend on temperature near the thermal transitions of the proteins. Significant differences in the temperature stability of Staphylococcal nuclease and the mutant exist; these proteins show characteristic thermal transition temperatures (Tm) of 51 and 30 degrees C, respectively, at pH 7. The temperature dependence of the intensity decay data are shown to be consistent with a two-state unfolding model. For both proteins, the longer rotational correlation time, due to overall rotational diffusion, decreases dramatically at the transition temperature, and the amplitude of the shorter correlation time increases, indicating increased segmental motions of the single tryptophan residue. The mutant protein appears to have a slightly larger overall rotational correlation time and to show slightly more segmental motion of its Trp than is the case for the wild-type protein.

Amino Acid Sequence↗
PubMed1991

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids.

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).

Acrylamides↗
PubMed1990

Fluorescence studies with potato carboxypeptidase inhibitor.

Potato carboxypeptidase inhibitor (CPI) is a 39-residue globular protein whose X-ray structure is known. The protein's two tryptophan residues (W22 and W28) appear to be on the surface in the crystal structure. The fluorescence spectrum of CPI has a maximum at 344 nm. Acrylamide solute quenching yields an upward curving Stern-Volmer plot with KSV approximately 9 M-1. KI quenching yields a linear plot with KSV approximately 5.5 M-1. These studies indicate that emission occurs from a solvent exposed residue(s). Fluorescence lifetime measurements were fitted to a biexponential decay law with tau 1 = 0.9 ns. f1 = 0.22 and tau 2 = 3.9 ns. Anisotropy decay data were described by a single rotational correlation time of 1.2 ns. This value is somewhat small for the global rotation of a protein of this size. The low temperature phosphorescence of CPI shows a biexponential decay, with a rapidly decaying 0.5-1.0 second component. The triplet state of at least one of the two tryptophan residues must be perturbed by interaction with nearby disulfide bonds.

Acrylamides↗
PubMed1990

Fluorescence studies with malate dehydrogenase from Bradyrhizobium japonicum 3I1B-143 bacteroids: a two-tryptophan containing protein.

A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate.

Kinetics↗
PubMed1989

Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine.

The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamides↗
PubMed1989

Fluorescence lifetime and solute quenching studies with the single tryptophan containing protein parvalbumin from codfish.

The fluorescence decay of cod parvalbumin (both its Ca2+-loaded and Ca2+-depleted forms) is found to be a nonexponential process. The decay data can be fitted either by a double-exponential decay law or by a distribution of decay times. To try to distinguish between the double-exponential and distribution fits, we have collected frequency domain and steady-state fluorescence data as a function of temperature and concentration of the quencher acrylamide. We argue that the correct decay law (i.e., double exponential or distribution) must be consistent with all the data collected as a function of temperature and quencher concentrations. We employ a global analysis procedure to simultaneously fit multiple data sets that are linked by an Arrhenius or Stern-Volmer relationship. For the Ca2+-loaded form of parvalbumin, the distribution model provides a consistent and reasonable fit for all of the frequency domain and steady-state data. The double-exponential model requires more fitting parameters, and some of these assume unreasonable values when this model is fitted to all of the data. For the Ca2+-depleted form of the protein, it is not clear whether the double-exponential or distribution model is superior. For our steady-state solute quenching studies we present a novel analysis in terms of a distribution of quenching constants.

Acrylamide↗
PubMed1989

Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures.

Binding Sites↗