Gediminas Vidugiris - Academia.edu (original) (raw)

Papers by Gediminas Vidugiris

たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融... more たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融合タンパク質をコード する1つのコンストラクトをトランジェントまたはステイブルなトラン スフェクションにより細胞内に導入します。現在、HaloTag 融合タン パク質の作成用のベクター3種(HaloTag pHT2 Vector、pFC8A、 pFC8K(HaloTag)CMV Flexi Vector)が利用できます。次に、細胞 透過性のリガンドを使用する場合、細胞を適切なリガンドとともに 5-60分間インキュベーションします。リガンドは細胞膜を透過し、 HaloTag 融合タンパク質に共有結合します。未結合のリガンドを洗浄 により除去した後、以降のアプリケーションに用います。細胞非透過 性のリガンドあるいはHaloLink を選択した場合、HaloTag リガンド とのインキュベーションを行う前に細胞を溶解します。図1に HaloTag TMR Ligand で標識したp65HaloTag 融合タンパク質を発 現する固定細胞のイメージを示します。 HaloTag Interchangeable Labeling Technologyの構成内容 HaloTag タンパク質は、原核生物 ヒドロラーゼに由来し、 HaloTag Ligandとの共有結合を形成させるために遺伝子組換えを行っ たタンパク質です(図2)。この33kDaの単量体タンパク質とのN末端あ るいはC末端融合体は様々な細胞種で効率よく発現させることができま す。HaloTag タンパク質は真核生物には存在しないので、高い標識特 異性を示します。HaloTag pHT2 Vectorには、多くの細胞種において 強力かつ構成的な発現を起こすCMVエンハンサー/プロモーター、5'ドナースプライスサイトの使用を最低限に抑えるキメラ・イントロン、 in vitro転写/翻訳システムに使用するT7プロモーター、HaloTag タン パク質コード配列そしてSV40 lateポリアデニル化シグナルが含まれて います(3)。上記の特長に加え、pFC8A および pFC8K(HaloTag) CMV Flexi Ve...

Journal of Fluorescence, Mar 1, 1998

The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutant... more The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutants of the trp repressor protein was characterized in both the native and the denatured states. These mutants belong to a particular class, called superrepressors, as their phenotype, when expressed in vivo, is to repress transcription at lower concentrations of the corepressor, tryptophan. It has been demonstrated previously that these mutations result in distinct and profound modifications of the structural and dynamic properties of the protein [Reedstrom and Royer (1995)J.

Bioelectrochemistry and Bioenergetics, Aug 1, 1989

An experimental study of adsorption processes involving proteins and polypeptides at the ekctronw... more An experimental study of adsorption processes involving proteins and polypeptides at the ekctronwnductive materiaI/eIect.roIyte interface is presented. The application of electrochemical and optical methods enabled us to determine in each case the limiting ackorption r,tep and the structure of the proteins at the interface with respect to the surface and protein properties and the electrolyte wmposi-tiOll.

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this... more Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for highthroughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1-5 pmol/sample), broad linear range (0.1-100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z′ = 0.6-0.9) and could be multiplexed with a realtime viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

Journal of Biological Chemistry, Dec 1, 1993

In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding... more In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding or changes in quaternary state are manifested at the heme as reflected in resonance Raman spectral changes involving the iron-proximal histidine stretching mode. No such changes are observed for Lumbricus terrestris hemoglobin. The iron-histidine stretching mode and the porphyrin breathing motion in the deoxy-, oxy-, or CO-photodissociated forms of Lumbricus hemoglobin and human hemoglobin A (pH 7.0 and 9.2, the latter to effect Lumbricus hemoglobin subunit dissociation) were studied using pulsed (10 ns) light at 435 nm. In contrast to that observed for hemoglobin A, a comparison of the spectra of the deoxy and photoproduct forms of Lumbricus hemoglobin reveal minimal differences in the region of the iron-histidine and the pi electron distribution in the heme moiety. The spectral frequencies are similar to that observed in R-state vertebrate hemoglobins. Such average behavior of the approximately 192 hemes present in Lumbricus hemoglobin is more analogous to the Raman spectral properties observed in myoglobin.

ACS Chemical Biology, Sep 22, 2011

Bioelectrochemistry and Bioenergetics, Jun 1, 1986

Reversible redox conversions of lactoperoxidase, ceruloplasmin and alkaline phosphatase disulfide... more Reversible redox conversions of lactoperoxidase, ceruloplasmin and alkaline phosphatase disulfide bonds proceed in phosphate buffer solutions on the d.m.e. Irreversibly adsorbed and denaturated enzyme molecules take part in the process. Enzymes are also adsorbed irreversibly on the d.m.e. from potassium chloride solutions, but the redox conversion rates of disulfide bonds are considerably lower, due to the specific adsorption of chloride ions on the mercury electrode.

Biochemistry, 1996

Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease fo... more Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to-glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42.

Assay and Drug Development Technologies, Oct 1, 2015

Real-time continuous monitoring of cellular processes offers distinct advantages over traditional... more Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time-and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z 0 = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

Biochimica Et Biophysica Acta - Proteins And Proteomics, Apr 1, 1994

Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus te... more Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus terrestris hemoglobin. Since tryptophan intrinsic fluorescence has been used as a reporter group to study Lumbricus hemoglobin, it is of interest to study dissociation perturbed properties of the tryptophan residues. Shifts in the fluorescence emission maximum to longer wavelengths upon dissociation at pH 9.2 suggest that tryptophans buried at the subunit interface(s) become more exposed. Fluorescence lifetime and quenching studies are employed in this present investigation as a means to confirm the location of tryptophan residues at the subunit interfaces. Acrylamide titration (to 2.5 M) indicate only a fraction of the residues can be quenched at either pH. At pH 7.0, the Stern-Volmer plot has downward curvature, while at pH 9.2 there is slight upward curvature, again indicating a change in environment. The intrinsic fluorescence decay requires at least four exponentials at both pHs. The mean fluorescence lifetime of CO Lumbricus hemoglobin increases from 1.1 ns at pH 7 to 3.3 ns at pH 9.2. The lifetime data can be further interpreted as a decrease in the quenching of residues with a = 30 ps lifetime, and a concomitant increase in the longer lifetime components. This is consistent with interface tryptophans becoming exposed to solvent upon dissociation, and loss of quenching by intersubunit hemes. The overall results suggest that in the dodecamer, most of the tryptophans are located in a hydrophobic environment, not all of which are located at the subunit interface.

Journal of electroanalytical chemistry and interfacial electrochemistry, Sep 1, 1988

In the presence of amino acids and proteins the cathodic reduction of AgCl in 0.1 M KC1 proceeds ... more In the presence of amino acids and proteins the cathodic reduction of AgCl in 0.1 M KC1 proceeds in two steps. The coordination number for amino acids is close to unity. Ceruloplasmin, peroxidase, methemoglobin, albumin and alkaline phosphatase molecules make a complex with 1.5-3 silver ions. Complex formation involves side groups of amino acids which contain hetero atoms. The constants of amino acid complex formation increase with increasing pK, values of the side groups. Protein complexation occurs mainly through lysine residues.

Journal of Magnetic Resonance, Sep 1, 2003

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening... more The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

Journal of Biological Chemistry, 1998

たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融... more たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融合タンパク質をコード する1つのコンストラクトをトランジェントまたはステイブルなトラン スフェクションにより細胞内に導入します。現在、HaloTag 融合タン パク質の作成用のベクター3種(HaloTag pHT2 Vector、pFC8A、 pFC8K(HaloTag)CMV Flexi Vector)が利用できます。次に、細胞 透過性のリガンドを使用する場合、細胞を適切なリガンドとともに 5-60分間インキュベーションします。リガンドは細胞膜を透過し、 HaloTag 融合タンパク質に共有結合します。未結合のリガンドを洗浄 により除去した後、以降のアプリケーションに用います。細胞非透過 性のリガンドあるいはHaloLink を選択した場合、HaloTag リガンド とのインキュベーションを行う前に細胞を溶解します。図1に HaloTag TMR Ligand で標識したp65HaloTag 融合タンパク質を発 現する固定細胞のイメージを示します。 HaloTag Interchangeable Labeling Technologyの構成内容 HaloTag タンパク質は、原核生物 ヒドロラーゼに由来し、 HaloTag Ligandとの共有結合を形成させるために遺伝子組換えを行っ たタンパク質です(図2)。この33kDaの単量体タンパク質とのN末端あ るいはC末端融合体は様々な細胞種で効率よく発現させることができま す。HaloTag タンパク質は真核生物には存在しないので、高い標識特 異性を示します。HaloTag pHT2 Vectorには、多くの細胞種において 強力かつ構成的な発現を起こすCMVエンハンサー/プロモーター、5'ドナースプライスサイトの使用を最低限に抑えるキメラ・イントロン、 in vitro転写/翻訳システムに使用するT7プロモーター、HaloTag タン パク質コード配列そしてSV40 lateポリアデニル化シグナルが含まれて います(3)。上記の特長に加え、pFC8A および pFC8K(HaloTag) CMV Flexi Ve...

Cancer Research, 2017

The critical importance of autophagy in cell health and its proposed role in disease-relevant bio... more The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing ...

SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2016

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this... more Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1–5 pmol/sample), broad linear range (0.1–100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor c...

Current chemical genomics, 2012

Our fundamental understanding of proteins and their biological significance has been enhanced by ... more Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenab...

たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融... more たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融合タンパク質をコード する1つのコンストラクトをトランジェントまたはステイブルなトラン スフェクションにより細胞内に導入します。現在、HaloTag 融合タン パク質の作成用のベクター3種(HaloTag pHT2 Vector、pFC8A、 pFC8K(HaloTag)CMV Flexi Vector)が利用できます。次に、細胞 透過性のリガンドを使用する場合、細胞を適切なリガンドとともに 5-60分間インキュベーションします。リガンドは細胞膜を透過し、 HaloTag 融合タンパク質に共有結合します。未結合のリガンドを洗浄 により除去した後、以降のアプリケーションに用います。細胞非透過 性のリガンドあるいはHaloLink を選択した場合、HaloTag リガンド とのインキュベーションを行う前に細胞を溶解します。図1に HaloTag TMR Ligand で標識したp65HaloTag 融合タンパク質を発 現する固定細胞のイメージを示します。 HaloTag Interchangeable Labeling Technologyの構成内容 HaloTag タンパク質は、原核生物 ヒドロラーゼに由来し、 HaloTag Ligandとの共有結合を形成させるために遺伝子組換えを行っ たタンパク質です(図2)。この33kDaの単量体タンパク質とのN末端あ るいはC末端融合体は様々な細胞種で効率よく発現させることができま す。HaloTag タンパク質は真核生物には存在しないので、高い標識特 異性を示します。HaloTag pHT2 Vectorには、多くの細胞種において 強力かつ構成的な発現を起こすCMVエンハンサー/プロモーター、5'ドナースプライスサイトの使用を最低限に抑えるキメラ・イントロン、 in vitro転写/翻訳システムに使用するT7プロモーター、HaloTag タン パク質コード配列そしてSV40 lateポリアデニル化シグナルが含まれて います(3)。上記の特長に加え、pFC8A および pFC8K(HaloTag) CMV Flexi Ve...

Journal of Fluorescence, Mar 1, 1998

The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutant... more The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutants of the trp repressor protein was characterized in both the native and the denatured states. These mutants belong to a particular class, called superrepressors, as their phenotype, when expressed in vivo, is to repress transcription at lower concentrations of the corepressor, tryptophan. It has been demonstrated previously that these mutations result in distinct and profound modifications of the structural and dynamic properties of the protein [Reedstrom and Royer (1995)J.

Bioelectrochemistry and Bioenergetics, Aug 1, 1989

An experimental study of adsorption processes involving proteins and polypeptides at the ekctronw... more An experimental study of adsorption processes involving proteins and polypeptides at the ekctronwnductive materiaI/eIect.roIyte interface is presented. The application of electrochemical and optical methods enabled us to determine in each case the limiting ackorption r,tep and the structure of the proteins at the interface with respect to the surface and protein properties and the electrolyte wmposi-tiOll.

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this... more Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for highthroughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1-5 pmol/sample), broad linear range (0.1-100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z′ = 0.6-0.9) and could be multiplexed with a realtime viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

Journal of Biological Chemistry, Dec 1, 1993

In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding... more In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding or changes in quaternary state are manifested at the heme as reflected in resonance Raman spectral changes involving the iron-proximal histidine stretching mode. No such changes are observed for Lumbricus terrestris hemoglobin. The iron-histidine stretching mode and the porphyrin breathing motion in the deoxy-, oxy-, or CO-photodissociated forms of Lumbricus hemoglobin and human hemoglobin A (pH 7.0 and 9.2, the latter to effect Lumbricus hemoglobin subunit dissociation) were studied using pulsed (10 ns) light at 435 nm. In contrast to that observed for hemoglobin A, a comparison of the spectra of the deoxy and photoproduct forms of Lumbricus hemoglobin reveal minimal differences in the region of the iron-histidine and the pi electron distribution in the heme moiety. The spectral frequencies are similar to that observed in R-state vertebrate hemoglobins. Such average behavior of the approximately 192 hemes present in Lumbricus hemoglobin is more analogous to the Raman spectral properties observed in myoglobin.

ACS Chemical Biology, Sep 22, 2011

Bioelectrochemistry and Bioenergetics, Jun 1, 1986

Reversible redox conversions of lactoperoxidase, ceruloplasmin and alkaline phosphatase disulfide... more Reversible redox conversions of lactoperoxidase, ceruloplasmin and alkaline phosphatase disulfide bonds proceed in phosphate buffer solutions on the d.m.e. Irreversibly adsorbed and denaturated enzyme molecules take part in the process. Enzymes are also adsorbed irreversibly on the d.m.e. from potassium chloride solutions, but the redox conversion rates of disulfide bonds are considerably lower, due to the specific adsorption of chloride ions on the mercury electrode.

Biochemistry, 1996

Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease fo... more Our recently reported pressure-jump relaxation kinetics experiments on staphylococcal nuclease folding and unfolding [Vidugiris et al. (1995) Biochemistry 34, 4909] demonstrated that both transitions exhibit positive activation volumes, with that of folding being much larger than that of unfolding. Thus high pressure denatures proteins by slowing the rate of folding more than that of unfolding. In the present work, we take advantage of the very slow folding and unfolding rates under pressure to examine the kinetics and volume changes along the reaction coordinate for protein folding-unfolding for an interesting set of mutants of staphylococcal nuclease: P42G, P47G, P117G, and the double mutant, P47G+P117G. Previous studies have shown that replacement of an individual proline residue at position 42, 47, or 117 by glycine leads to paradoxical protein stabilization against denaturation by guanidine chloride, high temperature, or high pressure. In order to observe unfolding over an attainable pressure range, guanidine hydrochloride was employed. Within experimental error, the activation volumes and equilibrium volume changes were independent of the concentration of this denaturant and our analysis of the rate constants is consistent with the generally accepted hypothesis that this denaturant acts both by increasing the rate of unfolding and decreasing the rate of folding. We show that the stabilization resulting from each of the proline-to-glycine substitutions arises primarily from a decrease in the unfolding rate, and to a small degree, from an increase in the folding rate. The changes in rate constants upon proline-to-glycine substitution can be modeled in terms of small stabilization of the unfolded state, a greater stabilization of the transition state, and a still greater stabilization of the folded state. Although the rates were found to change for all of the mutants in the set, no changes greater than experimental error were found in the corresponding equilibrium volume changes and activation volumes for folding and unfolding. At low pressures (well below the onset of unfolding) the pressure-jump relaxation profiles for wild type proteins (both Foggi and V8) showed kinetic complexity. Although the effect was attenuated somewhat in pressure-jump profiles of one proline-to-glycine mutant (P42G), its persistence in data from all the mutants studied leads us to conclude that its origin is not cis/trans peptide bond isomerization at proline 117, 47, or 42.

Assay and Drug Development Technologies, Oct 1, 2015

Real-time continuous monitoring of cellular processes offers distinct advantages over traditional... more Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time-and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z 0 = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

Biochimica Et Biophysica Acta - Proteins And Proteomics, Apr 1, 1994

Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus te... more Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus terrestris hemoglobin. Since tryptophan intrinsic fluorescence has been used as a reporter group to study Lumbricus hemoglobin, it is of interest to study dissociation perturbed properties of the tryptophan residues. Shifts in the fluorescence emission maximum to longer wavelengths upon dissociation at pH 9.2 suggest that tryptophans buried at the subunit interface(s) become more exposed. Fluorescence lifetime and quenching studies are employed in this present investigation as a means to confirm the location of tryptophan residues at the subunit interfaces. Acrylamide titration (to 2.5 M) indicate only a fraction of the residues can be quenched at either pH. At pH 7.0, the Stern-Volmer plot has downward curvature, while at pH 9.2 there is slight upward curvature, again indicating a change in environment. The intrinsic fluorescence decay requires at least four exponentials at both pHs. The mean fluorescence lifetime of CO Lumbricus hemoglobin increases from 1.1 ns at pH 7 to 3.3 ns at pH 9.2. The lifetime data can be further interpreted as a decrease in the quenching of residues with a = 30 ps lifetime, and a concomitant increase in the longer lifetime components. This is consistent with interface tryptophans becoming exposed to solvent upon dissociation, and loss of quenching by intersubunit hemes. The overall results suggest that in the dodecamer, most of the tryptophans are located in a hydrophobic environment, not all of which are located at the subunit interface.

Journal of electroanalytical chemistry and interfacial electrochemistry, Sep 1, 1988

In the presence of amino acids and proteins the cathodic reduction of AgCl in 0.1 M KC1 proceeds ... more In the presence of amino acids and proteins the cathodic reduction of AgCl in 0.1 M KC1 proceeds in two steps. The coordination number for amino acids is close to unity. Ceruloplasmin, peroxidase, methemoglobin, albumin and alkaline phosphatase molecules make a complex with 1.5-3 silver ions. Complex formation involves side groups of amino acids which contain hetero atoms. The constants of amino acid complex formation increase with increasing pK, values of the side groups. Protein complexation occurs mainly through lysine residues.

Journal of Magnetic Resonance, Sep 1, 2003

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening... more The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

Journal of Biological Chemistry, 1998

たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融... more たった1つのDNAコンストラクトだけでHaloTag テクノロジーの多 様な機能性を利用することができます。HaloTag テクノロジーを用い て融合タンパク質を標識するには、HaloTag 融合タンパク質をコード する1つのコンストラクトをトランジェントまたはステイブルなトラン スフェクションにより細胞内に導入します。現在、HaloTag 融合タン パク質の作成用のベクター3種(HaloTag pHT2 Vector、pFC8A、 pFC8K(HaloTag)CMV Flexi Vector)が利用できます。次に、細胞 透過性のリガンドを使用する場合、細胞を適切なリガンドとともに 5-60分間インキュベーションします。リガンドは細胞膜を透過し、 HaloTag 融合タンパク質に共有結合します。未結合のリガンドを洗浄 により除去した後、以降のアプリケーションに用います。細胞非透過 性のリガンドあるいはHaloLink を選択した場合、HaloTag リガンド とのインキュベーションを行う前に細胞を溶解します。図1に HaloTag TMR Ligand で標識したp65HaloTag 融合タンパク質を発 現する固定細胞のイメージを示します。 HaloTag Interchangeable Labeling Technologyの構成内容 HaloTag タンパク質は、原核生物 ヒドロラーゼに由来し、 HaloTag Ligandとの共有結合を形成させるために遺伝子組換えを行っ たタンパク質です(図2)。この33kDaの単量体タンパク質とのN末端あ るいはC末端融合体は様々な細胞種で効率よく発現させることができま す。HaloTag タンパク質は真核生物には存在しないので、高い標識特 異性を示します。HaloTag pHT2 Vectorには、多くの細胞種において 強力かつ構成的な発現を起こすCMVエンハンサー/プロモーター、5'ドナースプライスサイトの使用を最低限に抑えるキメラ・イントロン、 in vitro転写/翻訳システムに使用するT7プロモーター、HaloTag タン パク質コード配列そしてSV40 lateポリアデニル化シグナルが含まれて います(3)。上記の特長に加え、pFC8A および pFC8K(HaloTag) CMV Flexi Ve...

Cancer Research, 2017

The critical importance of autophagy in cell health and its proposed role in disease-relevant bio... more The critical importance of autophagy in cell health and its proposed role in disease-relevant biology, including cancer, inflammation, and immunology, has increased the need for more effective assays to screen for agents that modulate autophagic activity. Here we utilize NanoLuc Binary Technology (NanoBiT) to develop a homogeneous plate-based assay to measure autophagic flux in cell culture models. In this approach, an exogenous LC3B (Atg8) fusion protein was tagged on its N-terminus with an 11 amino acid peptide (HiBiT) and stably expressed in mammalian cells, including U2OS and HEK293. After exposure to various treatment conditions, cellular levels of this novel autophagy reporter were determined by addition of a lytic detection reagent containing Large BiT (LgBiT). LgBiT rapidly associates with HiBiT in the cell lysate, producing a bright, luminescent enzyme in the presence of the furimazine substrate. The bright signal allows low levels of expression of the reporter, maximizing ...

SLAS DISCOVERY: Advancing the Science of Drug Discovery, 2016

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this... more Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1–5 pmol/sample), broad linear range (0.1–100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor c...

Current chemical genomics, 2012

Our fundamental understanding of proteins and their biological significance has been enhanced by ... more Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenab...