RESUMEN
Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at -20 °C and -80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.
RESUMEN
Various biochemical methods have been introduced to detect and characterize KRAS activity and interactions, from which the vast majority is based on luminescence detection in its varying forms. Among these methods, thermal stability assays, using luminophore-conjugated proteins or external environment sensing dyes, are widely used. In this chapter, we describe methods enabling KRAS stability monitoring in vitro, with an emphasis on ligand-induced stability. This chapter focuses mainly on luminescence-based techniques utilizing external dye molecules and fluorescence detection.
Asunto(s)
Luminiscencia , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas/química , Mediciones Luminiscentes , Colorantes Fluorescentes/químicaRESUMEN
Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.
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Aminoácidos , Proteínas , Estabilidad Proteica , Proteínas/química , Fluorometría/métodos , Bioensayo , Desnaturalización ProteicaRESUMEN
Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of â¼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.
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Hidrodinámica , Albúmina Sérica Bovina , Rastreo Diferencial de Calorimetría , Temperatura de Transición , Animales , BovinosRESUMEN
Many molecular chaperones act as holdases by binding hydrophobic regions of substrates to prevent aggregation. Therefore, measuring holdase activity is an amenable method to determine chaperone activity. The holdase function is reliably and easily achieved by monitoring the suppression of heat-induced aggregation of well-characterized model protein substrates. However, the standard assay format requires large amounts of protein and hence is not applicable to all proteins. Using DnaK from Escherichia coli and heat-induced aggregation of malate dehydrogenase, we describe a protocol for absorbance and fluorescence-based miniaturized versions of the standard aggregation suppression assay that are affordable and have wide application for low abundance holdases. The assay can be used for both fundamental characterization of holdase function in proteins and screening of inhibitors of holdase activity.
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Proteínas de Escherichia coli , Agregado de Proteínas , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismoRESUMEN
Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.
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Proteínas de Unión al GTP Monoméricas , Bioensayo , Rastreo Diferencial de Calorimetría , Fluorometría/métodos , Estabilidad ProteicaRESUMEN
Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.
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Factor 2 Eucariótico de Iniciación , Desplegamiento Proteico , Adenosina Trifosfato/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Ligandos , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.
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Colorantes Fluorescentes , Albúmina Sérica Bovina , Adenosina Trifosfato , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Geles , Sefarosa , Dodecil Sulfato de Sodio , Coloración y EtiquetadoRESUMEN
Thermal shift assay (TSA) is a widely used method in discovering potential compounds (e.g., ligands, inhibitors, and other additives) to the target protein for structural genomics and drug screening in both academia and industry. The presence of sensitive fluorescent dye enables to monitor thermal stability of protein and compounds affecting this stability. By using a conventional real-time PCR instrument, it is determined as a low-cost and high efficacy experiment applied to identify optimal conditions for ligand binds to protein. Fatty acid-binding proteins (FABPs) are small molecular proteins in transporting fatty acids and other lipophilic substances in physiological and pathological responses. This chapter presents a comprehensive workflow to monitor recombinant FABP-compound interactions for an initial screening for inhibitors using TSA with SYPRO Orange dye.
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Proteínas de Unión a Ácidos Grasos/metabolismo , Colorantes Fluorescentes/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Antineoplásicos/farmacología , Línea Celular , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/química , Humanos , Ligandos , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , Temperatura de TransiciónRESUMEN
The development of STAT protein-specific inhibitors has been the focus of a number of drug discovery programs. STAT activation occurs through phosphorylation at the STAT SH2 domain, resulting in dimerization, translocation to the nucleus, and transcription of proliferative genes. Due to the functional significance of the SH2 domain in mediating multiple components of the STAT signalling cascade, many libraries of inhibitors have been designed to target the SH2 domain. This has triggered the requirement for effective high-throughput screening platforms for analyzing binding by larger chemical libraries to STAT proteins. Herein, we present strategies for the development of a high-throughput thermal denaturation-based assay for identifying STAT inhibitors as well as high-yielding recombinant expression and purification of untagged STAT1, STAT3, and STAT5 proteins. This assay reports changes in the fluorescence of a labelled peptide bound to the STAT protein as a function of increasing temperature. STAT inhibitors which displace the labelled peptide elicit a change in the melt profile, which is quantitatively determined as a change in the area under the curve. This assay offers an alternative, but complimentary, high-throughput screening strategy for identifying new inhibitors of STAT proteins as well as characterizing further, the mode of inhibition by existing libraries of compounds.
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Dominios Homologos src , Descubrimiento de Drogas , Fosforilación , Factores de Transcripción STAT , Transducción de Señal , Bibliotecas de Moléculas PequeñasRESUMEN
Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing - the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3' non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein.
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Proteínas de Unión al ADN , Escherichia coli/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
STAT5B, a ubiquitious transcription factor, has been implicated in the onset and progression of several cancers. Since the inhibition of STAT activity holds significant therapeutic potential, there is a need to develop high-throughput biophysical screening platforms to rapidly identify high affinity binders of STATs. Biophysical assays would benefit from the efficient and cost-effective production of high purity, full-length STAT proteins. Herein, we have sampled a large region of protein expression and purification space that has substantially increased recombinant STAT5B protein yields from Escherichia coli. The identity of STAT5B was confirmed by Western blotting analysis, while the results of a fluorescence polarization assay indicated that the purified protein is correctly folded and functional. A thermal shift assay was employed to assess the effect of various osmolytes on the stability of the protein. The protein expression conditions identified in this study allowed for more efficient and higher recovery of soluble STAT5B protein, which will enable a broad range of biophysical studies and facilitate high-throughput STAT5B drug screening.
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Escherichia coli/metabolismo , Expresión Génica , Factor de Transcripción STAT5 , Escherichia coli/genética , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factor de Transcripción STAT5/biosíntesis , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/aislamiento & purificación , SolubilidadRESUMEN
Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.
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Alanina-ARNt Ligasa/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Histidina-ARNt Ligasa/química , ARN de Transferencia Aminoácido-Específico/metabolismo , Treonina-ARNt Ligasa/química , Alanina-ARNt Ligasa/antagonistas & inhibidores , Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Benzopiranos/química , Inhibidores Enzimáticos/química , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Fluorometría/métodos , Histidina-ARNt Ligasa/antagonistas & inhibidores , Histidina-ARNt Ligasa/genética , Histidina-ARNt Ligasa/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Transición de Fase , Unión Proteica , Desplegamiento Proteico , ARN de Transferencia Aminoácido-Específico/genética , Especificidad por Sustrato , Treonina-ARNt Ligasa/antagonistas & inhibidores , Treonina-ARNt Ligasa/genética , Treonina-ARNt Ligasa/metabolismo , Aminoacilación de ARN de TransferenciaRESUMEN
Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type-2 diabetes. Amyloid sensitive dyes, particularly thioflavin-T, are widely used to detect ex-vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self-assembly. We show that the dye SYPRO-orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type-2 diabetes. No fluorescence enhancement is observed in the presence of pre-fibrillar species or in the presence of non-amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO-orange fluorescence and match the time course determined with thioflavin-T assays. Thus, SYPRO-orange offers an alternative to thioflavin-T assays of amylin amyloid formation. The implications for the interpretation of SYPRO-orange-based assays of protein stability and protein-ligand interactions are discussed.
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Amiloide/química , Colorantes Fluorescentes/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Benzotiazoles , Humanos , Cinética , Tiazoles/químicaRESUMEN
N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15µg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.
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Antiinfecciosos/farmacología , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/enzimología , Inhibidores Enzimáticos/farmacología , Antiinfecciosos/química , Inhibidores Enzimáticos/química , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1µM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner.
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Proteínas de Unión al ADN/química , Herpesvirus Humano 1/química , Temperatura de Transición/efectos de los fármacos , Ligandos , Estabilidad Proteica/efectos de los fármacosRESUMEN
Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro.
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Histonas/química , Microscopía Fluorescente/métodos , Nucleosomas/química , Histonas/metabolismo , Modelos Químicos , Modelos Moleculares , Nucleosomas/metabolismo , TemperaturaRESUMEN
A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT-PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins.