Biomedical subjects

J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 145 records · Page 8Linked to original sources

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

Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase.

The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence of Ca(2+)-independent reactions, such as enzyme phosphorylation with Pi in the reverse direction of the catalytic cycle. This reaction is not inhibited by Ca2+ in the labeled enzyme, while it is inhibited in the native enzyme. The NCD4 reaction which is involved in functional inactivation occurs in the membrane-bound portion of the ATPase. Sodium dodecyl sulfate solubilization of hydrophobic peptides, electrophoresis, and microsequencing of transblotted electrophoretic bands revealed that the fluorescent NCD4 label resides in a segment of tryptic fragment A1, intervening between Glu231 and Glu309. This segment includes two transmembrane helices, and does not include the domain involved in the phosphoryl transfer reaction during catalytic activity. This specific labeling does not occur when the NCD4 derivatization procedure is carried out in the presence of Ca2+ concentrations that also prevent functional inactivation. Fluorescence characterization by steady state and intensity decay measurements shows only negligible energy transfer between the NCD4 label and fluorescein isothiocyanate label of Lys515, indicating that the NCD4 label is unlikely to reside within the extramembranous region of the ATPase. On the other hand, the fluorescence emission of intrinsic tryptophan residues clustered within or near the transmembrane region of the ATPase, is distinctly affected by NCD4 label specifically bound to the ATPase, and NCD4 label nonspecifically bound to the sarcoplasmic reticulum membrane. The combined sequencing and spectroscopic observations indicate that derivatization with NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with specific calcium binding. This is in agreement with experiments (Clarke et al., 1989) demonstrating that mutations of any of six amino acids within the transmembrane region of the ATPase interfere with enzyme activation by Ca2+.

Amino Acid Sequence↗

PubMed1991

Distance distributions and anisotropy decays of troponin C and its complex with troponin I.

We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of the two-domain protein, respectively. We developed a procedure to correct for the fraction of the sample that was incompletely labeled with the acceptor independent of chemical data. At pH 7.5 and in the presence of Mg2+, the mean distance was near 15 A with a half-width of the distribution of 15 A; when Mg2+ was replaced by Ca2+, the mean distance increased to 22 A with a decrease in the half-width by 4 A. Similar but less pronounced differences in the mean distance and half-width between samples containing Mg2+ and Ca2+ were also observed with troponin C complexed to troponin I. The results suggest that the conformation of troponin C is altered by Ca2+ binding to the Ca(2+)-specific sites and displacing bound Mg2+ at the Ca2+/Mg2+ sites. This alteration may play an important role in Ca2+ signaling in muscle. At pH 7.5, the anisotropy decays of the donor-labeled troponin C showed two components, with the long rotational correlation time (12 ns) reflecting the overall motion of the protein. When the pH was lowered from 7.5 to 5.2, the mean distribution distance of apotroponin C increased from 22 to 32 A and the half-width decreased by a factor of 2 from 13 to 7 A. The long correlation time of apotroponin C increased to 19 ns at the acidic pH. These results are discussed in terms of a model in which skeletal troponin C is a dimer at low pH and enable comparison of the solution conformation of the protein at neutral pH with a crystal structure obtained at pH 5.2. While the conformation of the monomeric unit of troponin C dimer at pH 5.2 is extended and consistent with the crystal structure, the conformation at neutral pH is likely more compact than the crystal structure predicts.

Animals↗

PubMed1991

Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching.

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.

Acrylamide↗

PubMed1991

A 4-GHz frequency-domain fluorometer with internal microchannel plate photomultiplier cross-correlation.

We have developed and tested a multifrequency phase/modulation fluorometer based on the Hamamatsu Model R2024U gatable microchannel plate photomultiplier (MCP-PMT), using internal MCP-PMT cross-correlation. This internal mixing is accomplished by biasing and modulating the gating mesh which is located 0.2 mm behind the photocathode. Near the photocathode center, no high-frequency photocurrent modulation was achieved. Within a circular area near the photocathode edge, however, the R2024U allows accurate phase shift and demodulation measurements up to at least 4.5 GHz, the frequency limit of our PMT-modulation amplifier. By mixing immediately after the photocathode, there is no decrease in the time resolution due to transit time spread, and the MCP has to process only low-frequency signals. This means no low-level high-frequency signal voltages have to be handled in this fluorometer, and the problems of RF shielding become much less critical. Also, the effective output impedance of the PMT has been increased, resulting in a 43-dB increase in the PMT output signal power. In principle, more MCPs could be built into the PMT, allowing an improved fluorescence detection limit. We have used the method of reference fluorophores in order to compensate for pronounced PMT color effects, a wavelength-dependent modulation, and a wavelength-dependent time shift. No color correction is required in the case of time-dependent depolarization. The performance of the instrument was verified by measurements of the intensity decay of perylene, which showed a single-exponential decay, and by measurements of the decay of tryptophan in water, which showed a double-exponential decay, as expected.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence↗

PubMed1991

Correction for incomplete labeling in the measurement of distance distributions by frequency-domain fluorometry.

Measurements of time-resolved fluorescence are now being used to recover conformational distributions of biological macromolecules. The fluorescence data of the donor are easily corrupted by incomplete labeling of the macromolecules by the acceptor. In the present paper we describe a general procedure to correct for incomplete acceptor labeling in the determination of distance distributions from frequency-domain measurements of the donor fluorescence decay kinetics. The method can also be used to determine the extent of acceptor labeling. Simulated data were used to determine the effect of incomplete labeling on resolution of the distance distribution and the effect on the recovered distributions if one fails to account for incomplete labeling by the acceptor. The expressions and implemented algorithm were verified using known mixtures of donor-control and donor-acceptor pair molecules, which simulated the presence of a donor population lacking the acceptor. Finally, we present data on the distance distributions between two labeled sites in myosin S1 (Cys-697 to Cys-707) where it was not possible to obtain complete labeling of the acceptor site.

Algorithms↗

PubMed1991

Anisotropy decays of indole, melittin monomer and melittin tetramer by frequency-domain fluorometry and multi-wavelength global analysis.

We used frequency-domain fluorescence spectroscopy to measure the fluorescence lifetime and anisotropy decays of indole in propylene glycol, and of the tryptophan emission of melittin monomer and tetramer in water solutions at 5 degrees C. We obtained an increase in resolution of the anisotropy decays by using multiple excitation wavelengths, chosen to provide a range of fundamental anisotropy values. The multi-excitation wavelength anisotropy decays were analyzed globally to recover a single set of correlation times with wavelength-dependent anisotropy amplitudes. Simulated data and kappaR2 surfaces are shown to reveal the effect of multi-wavelength data on the resolution of complex anisotropy decays. For both indole and melittin, the anisotropy decays are heterogeneous and require two correlation times to fit the frequency-domain data. For indole in propylene glycol at 5 degrees C we recovered correlation times of 0.59 and 4.10 ns, which appear to be characteristic of the rigid and asymmetric indole molecule. For melittin monomer the correlation times were 0.13 and 1.75 ns, and for melittin tetramer 0.12 and 3.96 ns. The shorter and longer correlation times of melittin are due to segmental motions and overall rotational diffusion of the polypeptide.

Journal Article↗

PubMed1991

Intensity and anisotropy decays of the tyrosine calmodulin proteolytic fragments, as studied by GHz frequency-domain fluorescence.

Frequency-domain fluorescence measurements to 2 GHz were able to recover and account for essentially all of the intrinsic tyrosine anisotropy of calmodulin and its proteolytic fragments containing one or two tyrosine residues. Low-temperature measurements have detected a very rapid initial anisotropy decay in the 2-tyrosine species which may be attributed to radiationless energy transfer between the two tyrosines. The observed values of the rotational correlation times indicate that both tyrosines of calmodulin possess considerable mobility, which decreases in the presence of Ca2+ and at low temperatures.

Calmodulin↗

PubMed1991

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry.

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.

Mathematics↗

PubMed1991

Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry.

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.

Cysteine↗

PubMed1991

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy.

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.

Biophysical Phenomena↗

PubMed1990

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra.

We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.

Fluorescent Dyes↗

PubMed1990

Influence of end-to-end diffusion on intramolecular energy transfer as observed by frequency-domain fluorometry.

We investigated the influence of end-to-end diffusion on intramolecular energy transfer between a naphthalene donor and dansyl acceptor linked by polymethylene chain. A range of viscosities from 0.6 to 200 cP were obtained using propylene glycol at different temperatures (0-80 degrees C) and methanol at 20 degrees C. The intensity decays of naphthalene were measured in the frequency domain. Several theoretical models, including distance distributions, were used to fit the data. The results indicate that end-to-end diffusion of flexible donor-acceptor pairs can be detected and quantified using frequency-domain fluorometry, even in the presence of a distribution of donor-to-acceptor distances.

Diffusion↗

PubMed1990

Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer.

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.

Melitten↗

PubMed1989

Characterization of the tryptophan fluorescence from sarcoplasmic reticulum adenosinetriphosphatase by frequency-domain fluorescence spectroscopy.

We examined the tryptophan decay kinetics of sarcoplasmic reticulum Ca2+-ATPase using frequency-domain fluorescence. Consistent with earlier reports on steady-state fluorescence intensity, our intensity decays reveal a reproducible and statistically significant 2% increase in the mean decay time due to calcium binding to specific sites involved in enzyme activation. This Ca2+ effect could not be eliminated with acrylamide quenching, which suggests a global effect of calcium on the Ca2+-ATPase, as opposed to a specific effect on a single water-accessible tryptophan residue. The tryptophan anisotropy decays indicate substantial rapid loss of anisotropy, which can be the result of either intramolecular energy transfer or a change in segmental flexibility of the ATPase protein. Energy transfer from tryptophan to TNP-ATP in the nucleotide binding domain, or to IEADANS on Cys-670 and -674, indicates that most tryptophan residues are 30 A or further away from these sites and that this distance is not decreased by Ca2+. In light of known structural features of the Ca2+-ATPase, the tryptophan fluorescence changes are attributed to stabilization of clustered transmembrane helices resulting from calcium binding.

Adenosine Triphosphate↗

PubMed1989

Decay time distribution analysis of Yt-base in benzene-methanol mixtures.

Frequency-domain fluorometry was used to measure intensity decays of synthetic Yt-base in mixtures of benzene-methanol at 20 degrees C. Multiexponential analysis shows that the decay of Yt-base fluorescence in benzene and methanol can be well fitted to a single-exponential model with tau = 9.67 ns and 6.25 ns respectively. In mixtures of benzene-methanol the decays became heterogeneous, and the maximum of heterogeneity observed was in a mixture containing 6% methanol. Since we expected a distribution of Yt-base solvation states in the solvent mixtures, and because the decay times of Yt-base are sensitive to solvent, we analyzed the data in terms of decay time distributions. The goodness-of-fit for the unimodal distribution model which has two floating parameters was equivalent to that found using the double exponential model with three floating parameters. The Lorentzian distribution model appears to provide a slightly superior fit relative to the Gaussian distribution model. These results suggest that the intensity decays of solvent-sensitive fluorophores in mixed solvents are described by a distribution of decay times.

Benzene↗

PubMed1989

Resolution of two emission spectra for tryptophan using frequency-domain phase-modulation spectra.

We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.

Oxazoles↗

PubMed1988

Distance distributions in proteins recovered by using frequency-domain fluorometry. Applications to troponin I and its complex with troponin C.

We used resonance energy transfer to examine the distribution of distances between two sites on troponin I (TnI). The donor (D) was the single tryptophan residue at site 158 (Trp 158), and the acceptor (A) was cysteine 133 (Cys 133) which was labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IE). A distribution of D-A distances results in a distribution of donor decay times, which were resolved by using frequency-domain fluorometry. In the native state we recovered a relatively narrow distribution of D-A distances. The widths of the distance distributions were found to increase progressively and dramatically with increasing concentrations of guanidine hydrochloride. Binding of calcium-free troponin C (TnC) to troponin I did not alter the distance distribution. Addition of Ca2+ to the TnI.TnC complex resulted in a sharper distance distribution and protected against the guanidine hydrochloride induced increase in the width of the distance distribution. Additionally, the same distance distributions were recovered for native and denatured TnI when the Forster distance for energy transfer was decreased by acrylamide quenching. These results demonstrate that distance distributions can be recovered with good accuracy, to the extent of revealing modest changes due to binding of other components. This technique should have widespread applications in studies of protein folding.

Fluorometry↗