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J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 37 records · Page 2Linked to original sources
PubMed2000

A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin.

The molecular motor myosin is proposed to bind to actin and swing its light-chain binding region through a large angle to produce an approximately 10 nm step in motion coupled to changes in the nucleotide state at the active site. To date, however, direct dynamic measurements have largely failed to show changes of that magnitude. Here, we use a cysteine engineering approach to create a high resolution, FRET-based sensor that reports a large, approximately 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.

Adenosine Diphosphate↗
PubMed2000

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.

Archaeoglobus fulgidus↗
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

Pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius as probe to monitor the sodium concentrations in the blood.

We describe the isolation and characterization of a pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius. This protein appears to be a tetramer composed of four 55-kDa subunits. The intrinsic tryptophan fluorescence of this protein is quenched by approximately 20% upon binding sodium, which occurs with a dissociation constant near 15 mM. Importantly, the intrinsic fluorescence of this pyruvate kinase does not appear to be affected by potassium, magnesium, and calcium at the concentrations found in whole blood. It appears that this pyruvate kinase can provide the basis for a selective protein sensor for sodium with minimal interference from other cations.

Ammonium Sulfate↗
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

Fluorescence properties of rhodamine 800 in whole blood and plasma.

We have characterized the fluorescence spectral properties of rhodamine 800 (Rh800) in plasma and blood in order to test the possibility of making clinical fluorescence measurements in whole blood without separation steps. Rh800 was used because of its absorption at red/near-infrared wavelengths away from the absorption bands of hemoglobin. We utilized the front-face illumination and detection to minimize the effects of absorption and/or scatter during measurements. The presence of Rh800 was detected in plasma and blood using steady-state fluorescence measurements. Absorption due to hemoglobin reduced the Rh800 intensity from the blood. Fluorescence lifetime measurements in plasma and blood showed that it is possible to recover lifetime parameters of Rh800 in these media. We obtained mean lifetimes of 1.90 and 1.86 ns for Rh800 in plasma and blood, respectively. Using the recently described modulation sensing method, we quantified the concentrations of Rh800 in plasma and blood. Rh800 was detected at a concentration of as low as 2 microM in both media. High anisotropy values were obtained for Rh800 in plasma and blood using steady-state and anisotropy decay measurements, implying the tight binding of this probe to the contents of these media. This binding can be exploited to monitor the concentrations of different blood components using already existing or new red-emitting probes that will be specially designed to bind to these components with high specificity. To test this possibility of direct measurements in blood, we used Rh800 to monitor albumin in the presence of red blood cells. Increase in the polarization of Rh800 as the concentration of albumin was increased in the presence of the red cells showed the feasibility of such measurements.

Blood Chemical Analysis↗
PubMed2000

The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature.

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.

Archaeoglobus fulgidus↗
PubMed2000

Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments.

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-¿[(dimethylamino)phenyl]azo¿phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.

Acrylamide↗
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

Thermodynamics and dynamics of histidine-binding protein, the water-soluble receptor of histidine permease. Implications for the transport of high and low affinity ligands.

The bacterial histidine permease is a model system for ABC transporters (traffic ATPases). The water-soluble receptor of this permease, HisJ, binds L-histidine and L-arginine (tightly) and L-lysine and L-ornithine (less tightly) in the periplasm, interacts with the membrane-bound complex (HisQMP2) and induces its ATPase activity, which results in ligand translocation. HisJ is a two-domain protein; in the absence of ligand, the cleft between two domains is open and binding of substrate stabilizes the closed conformation. Surprisingly, various liganded HisJ forms display substantial differences in their physicochemical characteristics and capacity to induce the ATPase. This is due to either different effects of the individual ligands on the respective closed structures, or to different equilibria being reached for each ligand between the open liganded form and the closed liganded form [Wolf, A. , Lee, K.C., Kirsch, J.F. & Ames, G.F.-L. (1996) J. Biol. Chem. 271, 21243-21250]. In this work, time-resolved measurements of the decay of intrinsic HisJ fluorescence and of the decay of the anisotropy of the fluorescence, as well as the analysis of the steady-state near UV CD and fluorescence spectra, rule out the model in which the differences between liganded complexes reflect different equilibria. The decay of the anisotropy of the fluorescence shows that liganded complexes differ dramatically in their large-scale conformational dynamics. Differential scanning calorimetry (DSC) curves for the HisJ thermal unfolding are well described by a scheme of equilibrium two-state unfolding of two independent domains, which can be ascribed to the two-domain structure of HisJ. This is true both for apo-HisJ at various pH values, and for HisJ in the presence of its ligands at varying concentrations, at pH 8.3. The DSC and structural data suggest that all ligands interact more extensively with the larger domain. A qualitative model for the HisJ conformational dynamics employing the idea of a twisting movement of the domains is proposed, which explains the difference in the efficacy of the ATPase induction by the various liganded HisJ forms.

ATP-Binding Cassette Transporters↗
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↗
PubMed2000

On spectral relaxation in proteins.

During the past several years there has been debate about the origins of nonexponential intensity decays of intrinsic tryptophan (trp) fluorescence of proteins, especially for single tryptophan proteins (STP). In this review we summarize the data from diverse sources suggesting that time-dependent spectral relaxation is a ubiquitous feature of protein fluorescence. For most proteins, the observations from numerous laboratories have shown that for trp residues in proteins (1) the mean decay times increase with increasing observation wavelength; (2) decay associated spectra generally show longer decay times for the longer wavelength components; and (3) collisional quenching of proteins usually results in emission spectral shifts to shorter wavelengths. Additional evidence for spectral relaxation comes from the time-resolved emission spectra that usually shows time-dependent shifts to longer wavelengths. These overall observations are consistent with spectral relaxation in proteins occurring on a subnanosecond timescale. These results suggest that spectral relaxation is a significant if not dominant source of nonexponential decay in STP, and should be considered in any interpretation of nonexponential decay of intrinsic protein fluorescence.

Animals↗
PubMed1999

The fluorescence emission of the apo-glucose oxidase from Aspergillus niger as probe to estimate glucose concentrations.

We developed a new method of glucose sensing using an inactive form of glucose oxidase from Aspergillus niger. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-1-naphthalenesulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the K(D) of holo-glucose oxidase. These results suggest that apo-glucose oxidase can be used as a reversible nonconsuming sensor for glucose.

Anilino Naphthalenesulfonates↗
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↗