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I Gryczynski

Publications and source records attributed to I Gryczynski.

At least 19 recordsLinked to original sources
PubMed2000

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor.

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Animals↗
PubMed2000

A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.

Anilino Naphthalenesulfonates↗
PubMed2000

Calcium-induced flexibility changes in the troponin C-troponin I complex.

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.

Animals↗
PubMed2000

Time-resolved spectral observations of cadmium-enriched cadmium sulfide nanoparticles and the effects of DNA oligomer binding.

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS-Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.

Biopolymers↗
PubMed2000

Background suppression in frequency-domain fluorometry.

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.

Fluorescence↗
PubMed2000

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations.

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.

Animals↗
PubMed2000

Novel fluorescence sensing methods for high throughput screening.

We describe two new methods of fluorescence sensing for use in high throughput screening (HTS). Modulation sensing transforms analyte-dependent intensity changes into a change in the low-frequency modulation signal. Polarization sensing transforms an intensity change into a change in polarization. Both methods are internally calibrated by using a reference film immediately adjacent to the sample, which can be readily located on the HTS plate or on a nearby optical component and provides an intensity or polarization reference. Modulation sensing and polarization sensing were both shown useful for measurements of fluorophore concentrations, pH, or calcium concentrations in the wells of HTS plates. Sensing with a reference film provides the opportunity to internally reference HTS measurements without the need for additions to the sample. This approach can provide standardization for assays performed at different times.

Fluorescence Polarization↗
PubMed2000

End-to-end diffusion on the microsecond timescale measured with resonance energy transfer from a long-lifetime rhenium metal-ligand complex.

We measured the end-to-end diffusion coefficient of an alkyl chain-linked donor-acceptor pair using the time-resolved frequency-domain decay of the donor. The donor was a rhenium metal-ligand complex with a mean decay time ranging from 2.1 to 7.9 microseconds in the absence of the Texas red acceptor. The decay time was used to measure the donor-to-acceptor distance distribution and the mutual diffusion coefficient. Using this long lifetime donor, it was easily possible to determine a diffusion coefficient near 2 x 10(-8) cm2/s and diffusion coefficients as low as 1.3 x 10(-9) cm2/s were measurable. Such long lifetime donors should be valuable for measuring the flexing of peptides on the microsecond timescale, domain motions of proteins and lateral diffusion in membranes. The availability of microsecond decay time luminophores now allows luminescence spectroscopy to be useful generally for studies of microsecond dynamics of biological macromolecules.

Biopolymers↗
PubMed1999

The beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100 degrees C.

Enzymes from thermophilic organisms are stable and active at temperatures which rapidly denature mesophilic proteins. However, there is not yet a complete understanding of the structural basis of their thermostability and thermoactivity since for each protein there seems to exist special networks of interactions that make it stable under the desired conditions. Here we have investigated the activity and conformational dynamics above 100 degrees C of the beta-glycosidase isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. This has been made possible using a special stainless steel optical pressure cell which allowed us to perform enzyme assays and fluorescence measurements up to 160 degrees C without boiling the sample. The beta-glycosidase from S. solfataricus showed maximal activity at 125 degrees C. The time-resolved fluorescence studies showed that the intrinsic tryptophanyl fluorescence emission of the protein was represented by a bimodal distribution with Lorential shape and that temperature strongly affected the protein conformational dynamics. Remarkably, the tryptophan emission reveals that the indolic residues remain shielded from the solvent even at 125 degrees C, as shown by shielding from quenching and restricted tryptophan solubility. The relationship between enzyme activity and protein structural dynamics is discussed.

Algorithms↗
PubMed1999

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.

Fluorescence Polarization↗
PubMed1999

Polarization sensing with visual detection.

We describe a new approach to fluorescence sensing which relies on visual determination the polarization. The sensing device consists of a fluorescent probe, which changes intensity in responses to the analyte, and an oriented fluorescent film, which is not affected by the analyte. An emission filter is selected to observe the emission from both the film and the sensing fluorophore. Changes in the probe intensity result in changes in the polarization of the combined emission from the sensor and reference. The degree of polarization can be detected visually using a dual polarizer with adjacent sections oriented orthogonally to each other. The emission passing through the dual polarizer is viewed with a second analyzing polarizer. This analyzer is rotated manually to yield equal intensities from both sides of the dual polarizer. This approach was used to measure the concentration of RhB in intralipid and to measure pH using 6-carboxyfluorescein. The analyzer angle is typically accurate to 1 degree, providing pH values accurate to +/- 0.1 pH unit at the midpoint of the titration curve. We also describe a method of visual polarization sensing that does not require an oriented film and that can use the same fluorophore for the sample and reference. These approaches to visual sensing are generic and can be applied to a wide variety of analytes for which fluorescent probes are available. Importantly, the devices are simple, with the only electronic component being the light source.

Algorithms↗
PubMed1999

Anisotropy-based sensing with reference fluorophores.

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.

Anisotropy↗
PubMed1999

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

Bacterial Proteins↗
PubMed1999

Long-lifetime lipid rhenium metal-ligand complex for probing membrane dynamics on the microsecond timescale.

We report the luminescence and spectral properties of a phospholipid analogue containing a long-lifetime luminescent rhenium metal-ligand complex (MLC) covalently linked to the amino group of phosphatidyl ethanolamine. When incorporated into synthetic membranes, this lipid probe displays intensity decay times near 3 microseconds. Importantly, the probe displays highly polarized emission with a maximal fundamental anisotropy of 0.33. This probe is expected to have numerous applications for studies of microsecond diffusion and dynamics of membranes.

Diffusion↗
PubMed1999

Resonance energy transfer study using a rhenium metal-ligand lipid conjugate as the donor in a model membrane.

We measured steady state and time-resolved resonance energy transfer between donors and acceptors in model membranes. The donor was a long lifetime rhenium-lipid complex, which displayed a mean lifetime of 1 microsecond and lifetime components as long as 3 microseconds in the labeled DOPC membranes. The transfer efficiencies were found to be substantially larger than those predicted without consideration of lateral diffusion. The larger transfer efficiencies are consistent with a mutual lateral diffusion coefficient in the membrane near 2 x 10(-8) cm2/s. These results demonstrate that lateral diffusion in membranes can be detected with microsecond lipid probes.

Energy Transfer↗
PubMed1999

Polarization-based oxygen sensor.

A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.

Fluorescence Polarization↗
PubMed1999

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.

1-Butanol↗