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J R Knutson

Publications and source records attributed to J R Knutson.

At least 37 records · Page 2Linked to original sources
PubMed1992

Interference of diffusive light waves.

We examine interference effects resulting from the superposition of photon-density waves produced by coherently modulated light incident upon a turbid medium. Photon-diffusion theory is used to derive expressions for the ac magnitude and phase of the aggregate diffusive wave produced in full- and half-space volumes by two sources. Using a frequency-domain spectrometer operating at 410 MHz, we verify interference patterns predicted by the model in scattering samples having optical properties similar to those of skin tissue. Potential imaging applications of interfering diffusive waves are discussed in the context of the theoretical and experimental results.

Humans↗
PubMed1991

Interaction of influenza hemagglutinin amino-terminal peptide with phospholipid vesicles: a fluorescence study.

We have studied tryptophan fluorescence from a 20-residue synthetic peptide corresponding to the amino terminal of the HA2 subunit of the influenza virus hemagglutinin protein, a putative "fusion" peptide. Decay-associated spectra have been obtained at pH 7.4 and at pH 5 (the optimal pH for influenza virus fusion) in the presence and absence of liposomes. We demonstrate that a blue shift in the total steady-state fluorescence spectrum upon binding to liposomes is due to a movement in characteristic emission wavelength and increased lifetime of one of the resolved spectral components. In contrast, a further shift after lowering the pH is the product of a redistribution in the relative amplitudes of spectral components. Also, each decay component is quenched by spin-labels or anthroxyl groups normally located within the hydrocarbon interior of the membranes. Calculations are presented leading to an estimate of the distance of the tryptophan residue from the bilayer center, suggesting that the tryptophan residues are at or near the hydrocarbon-polar interface. No gross positional change was detected between pH values. Rotational depolarization is shown to be retarded by liposome binding, more so at low pH.

Amino Acid Sequence↗
PubMed1991

Fluorescence of tryptophan dipeptides: correlations with the rotamer model.

The multiexponential decay of tryptophan derivatives has previously been explained by the presence of rotamers having different fluorescence lifetimes, but it has been difficult to correlate rotamer structure and physical properties. New time-resolved and static data on dipeptides of the type Trp-X and X-Trp, where X is another aminoacyl residue, are consistent with the rotamer model and allow some correlations. That a dominant rotamer of Trp-X zwitterion has the -NH3+ group near the indole ring was inferred from absorption and fluorescence spectra, titrimetric determination of pKa values, photochemical hydrogen-deuterium-exchange experiments, decay-associated spectra, quantum yields, and decay kinetics. Analysis of the lifetime and quantum yield data for Trp dipeptides, especially X-Trp, suggests that static self-quenching is not uncommon. Highly quenched and weak components of the fluorescence do not contribute to the calculated mean lifetime, thus resulting in apparent static quenching. We propose the term quasi-static self-quenching (QSSQ) to distinguish this phenomenon from quenching due to ground-state formation of a dark complex. Mechanisms of quenching and the structure of statically quenched rotamers are discussed. The occurrence of QSSQ supports the idea that rotamers interconvert slowly. A major perceived deficiency of the rotamer model, namely, the apparent inability to predict reasonable rotamer populations from fluorescence decay data, may result from the presence of statically quenched species, which do not contribute to the fluorescence.

Dipeptides↗
PubMed1990

Steady-state fluorescence and time-resolved fluorescence monitor changes in tryptophan environment in arginase from Saccharomyces cerevisiae upon removal of catalytic and structural metal ions.

Yeast arginase is a trimeric protein of identical subunits, each containing three tryptophans. Time-resolved fluorescence and steady-state fluorescence were employed to monitor the effects of removing the weakly bound catalytic Mn2+ as well as the tightly bound structural Zn2+/Mn2+. Resolution of the total native emission spectrum into decay-associated spectra (DAS) yielded components with lifetimes of 0.1, 1.2, and 4.0 ns. Upon removal of the catalytic metal, the intensities increased approximately 20% while the lifetimes increased less than 10%, and the DAS were unchanged except in intensity. The two major components are well resolved, but the 0.1-ns term is small and dominated by scattered excitation. In contrast, removal of the structural metal increased decay times to 0.2, 1.8, and 5.3 ns. More important, both native DAS red-shifted and became indistinguishable. These data suggest that removal of the catalytic metal does little to change the microenvironments of the individual tryptophans while removal of the structural metal causes partial unfolding of the protein. The excitation spectra for the active and inactive trimers were resolved into their excitation DAS (IEDAS), suggesting ground-state heterogeneity of the fluorescent species. In contrast, the excitation spectra of arginase without the structural metal could not be resolved due to the indistinguishable DAS. The tryptophans are quenched by acrylamide but not by cesium or iodide. Global analysis of the acrylamide quenching data resulted in two quenching decay-associated spectra (QDAS) which correlated well with the DAS. Since the apoenzyme does not exhibit tryptophan accessibility to either positive or negative ionic quenchers, one must assume that the "unfolded" monomeric protein retains considerable tertiary structure.

Apoenzymes↗
PubMed1990

Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red.

Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.

Animals↗
PubMed1990

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I.

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.

Dithionitrobenzoic Acid↗
PubMed1988

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers.

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Förster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.

Chemical Phenomena↗
PubMed1987

Nanosecond time-resolved fluorescence measurements during protein denaturation.

A procedure is described by which the information available from nanosecond time-resolved fluorescence measurements can be used to study rates of reactions taking place on time scales of seconds to hours. A pulse fluorometer was modified so as to obtain a series of short sequential data collections which were rapidly stored on computer disk files. As an application of this new methodology, the unfolding of horse liver alcohol dehydrogenase under acid conditions was monitored by changes in the decay parameters of the intrinsic fluorescence. Although the individual decay curves each had relatively few counts (330 to 160 counts at the peak), a series of decay curves obtained as a function of time could be analyzed in terms of a biexponential function. It was found that the decrease in steady-state fluorescence could be explained most simply by a decrease in the amplitude associated with the longer of the two decay constants.

Alcohol Dehydrogenase↗
PubMed1987

Nanosecond time-resolved fluorescence kinetic studies of the 5,5'-dithiobis(2-nitrobenzoic acid) reaction with enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.

Enzyme I of the bacterial phosphotransferase system is a protein component which undergoes a temperature-dependent monomer/dimer equilibrium. Reaction of sulfhydryl residues with SH-specific reagents inhibits both activity and dimerization. There are four cysteine residues available in each subunit, one of which (Cys 502) is proximate to one of the two tryptophan residues (Trp 498). Previous studies revealed two major lifetimes and spectra, suggesting distinct environments for tryptophan. In this paper, we examine the dynamic quenching of tryptophanyl fluorescence that occurs when an energy transfer acceptor, thio-2-nitrobenzoic acid (TNB), is covalently attached to the sulfhydryl groups. More precisely, we have traced the recovery of nativelike fluorescence lifetime components (and the concomitant loss of "reduced lifetime" amplitudes) that accompanies TNB release. The course of lifetime changes seen when a reducing reagent removes the quencher may be sensitive to a variety of effects, including different SH affinities, different proximities to Trp, changing availability for dimerization, or conformational changes. The prospective value of separating each lifetime component from the mixture is illustrated.

Dithionitrobenzoic Acid↗
PubMed1986

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 1. Theory and applications.

Individual fluorescence spectra for species in a heterogeneous system can be determined by using differences between the rotational correlation times of those components. Each spectrum derived is associated with a particular fluorescence anisotropy decay function; hence, they are anisotropy decay associated spectra (ADAS). We have previously shown [Knutson, J. R., Walbridge, D. G., & Brand, L. (1982) Biochemistry 21, 4671-4679] that a system containing different decay functions for total intensity can be resolved into constituent decay-associated spectra. ADAS extends the technique into the realm of fluorescence polarization, making use of the often disparate Brownian rotations found in heterogeneous biochemical systems. In this paper, we present the basic theory for ADAS in various heterogeneous systems and then present an example of ADAS resolving a binary mixture of macromolecules into "fast-rotor" (smaller or more mobile) and "slow-rotor" (larger or less mobile) components. They correctly superimpose spectra taken for the unmixed components. In the companion paper [Davenport, L., Knutson, J. R., & Brand, L. (1986) Biochemistry (following paper in this issue)], a specific application to a problem of importance of lipid biochemistry--e.g., the origin of the membrane probe order parameter in lipid bilayers--is presented, demonstrating the role rotational heterogeneity may play in biochemical fluorescence.

Fluorescence Polarization↗
PubMed1986

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers.

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.

Dimyristoylphosphatidylcholine↗
PubMed1986

Excited-state proton transfer of equilenin and dihydroequilenin: interaction with bilayer vesicles.

The two-state excited-state proton-transfer process for d-equilenin [d-3-hydroxyestra-1,3,-5(10),6,8-pentaen-17-one] and dihydroequilenin is found to depend both on pH and on proton acceptor concentration. Both the protonated and deprotonated forms of the excited molecule are fluorescent. As is the case for 2-naphthol, the excited-state pKa (pKa*) is substantially lower than the ground-state pKa. Fluorescence decay studies have been performed as a function of emission wavelength in aqueous solutions at pH 6.9 in the presence of acetate anion (0.1 M). At this pH, both back-reaction from the excited-state and ground-state heterogeneity are minimal. A monoexponential decay is found in the blue region of the spectrum and a biexponential decay on the red edge. The lifetimes measured across both regions are constant, with a negative preexponential term, characteristic of an excited-state reaction, evident at longer wavelengths. Decay-associated spectra (DAS), the preexponential terms associated with the measured lifetimes, have been acquired for these aqueous solutions. Equilenin and dihydroequilenin are found to adsorb to dimyristoyllecithin (DML) vesicles. Rates for excited-state proton transfer are greatly reduced when dihydroequilenin adsorbs to vesicles. The accessibility of the bound probe to acetate as a proton acceptor depends on the cholesterol content of the vesicles.

17-Ketosteroids↗
PubMed1982

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase.

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.

Alcohol Dehydrogenase↗