Name: determination of protein-ligand interactions using differential scanning fluorimetry
Uploaded: 2014-09-13T15:00:00.000+00:00
Duration: 13 min 26 s
Description: Watch this Scientific Journal Video about Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry at JoVE.com

This work was funded by grant from the BBSRC (grant number BB/H019685/1 and BB/E527663/1) to the University of Exeter.

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The authors declare that they have nothing to disclose.

Все белки связываются с разной сродства, для широкого круга других молекул из простых ионов в других крупных макромолекул. Во многих случаях, белки связываются с малых партнеров молекул в рамках своей нормальной функции (например, киназа связывания с АТФ). Другие взаимодействия может быть не связан с функцией, но экспериментально полезны в качестве инструментов (например, малых молекул, которые стабилизируют белки улучшить успех кристаллизации, или оказания помощи в поддержании белки в растворе); в то время как малые молекулы, которые связываются с активными центрами и аллостерическими сайтами белков могут действовать как ингибиторы, так и модулируют активность ферментов. Есть широкий спектр методов, которые могут быть использованы для определения аффинности белков для молекул-партнеров. Изотермический титрования калориметрии 1 широко рассматривается как «золотой стандарт», так как он обеспечивает богатую информацию о реакциях, является этикетка бесплатно, и ограниченные возможности дляrtifacts эксперимента. Тем не менее, несмотря на недавние улучшения в чувствительности измерительных приборов и автоматизации экспериментальной установки, это все еще относительно дорогим с точки зрения потребностей в белке, в лучшем случае низкого и среднего пропускную способность, и лучше всего подходит для взаимодействия с умеренной высокое сродство (10 нм до 100 мкм K D) 2. Другие этикеток бесплатных методы, такие как поверхностного плазмонного резонанса или двухслойной интерферометрии 3 предложения более высокой производительности, и добились чувствительность для обнаружения молекул меньшего размера, как низкие, как 100 Da. Тем не менее, высокие пропускная инструменты для этих методов сравнительно дорого, оправданы только там, где будет постоянное пропускная соответствующих проектов, и так, вероятно, будут недоступны для многих академических лабораториях. Дифференциальная сканирующая флуориметрия (DSF, или thermofluor) был впервые описан в 2001 году 4 в качестве метода для обнаружения наркотиков. В этом метод, белки инкубировали с флуоресцентным красителем (первоначально были использованы нафталин-сульфоновой красители), который изменяет свое флуоресценции при связывании с гидрофобными участками белков. Образец белка с красителем затем нагревают, а также мониторинг флуоресценции по мере возрастания температуры. Разворачивание белка, а также воздействие гидрофобных частей белка, приводит к характерным рисунком в флуоресценции в зависимости от температуры (фиг.1А). Эксперимент может быть выполнена в небольших объемах в любом коммерческом количественной ПЦР инструмента, и таким образом, в одном эксперименте, большое количество образцов одновременно могут быть проверены (обычно 48, 96 или 384 образцов, в зависимости от модели прибора). Опыты, как правило, могут быть выполнены в вокруг часов, что обеспечивает возможность высокопроизводительного анализа образцов 5. Дальнейшее совершенствование методологии видели принятие красителей с лучшими спектральными свойствами 6,7 </вир&gt;, генетические методы для анализа данных, и предложил протоколы для первичного скрининга 8,9. Область применения метода была расширена, с особым акцентом на создание оптимальных условий для подготовки и хранения белков 10, и на выявлении потенциальных партнеров по связыванию, чтобы помочь кристаллизации 11. Относительно высокая пропускная способность метода, относительно низкая стоимость белком (~ 2 мкг на реакции), и применимость к изучению слабых связывающие молекулы сделал DSF собой ценный инструмент для фрагмента на основе разработки лекарств, особенно в академическом контексте 12-14. Несмотря на широкое применение DSF к изучению белок-лиганд, несколько исследований описано определение констант диссоциации этих исследований. Тем не менее, эти, как правило, производят подробные уравнения, описывающие развертывание белка, по многим параметрам, которые должны быть установлены в разреженных данных или в некоторых случаях еstimated 7,15-17. Эти методы имеют особое значение в сложных случаях, например, плотно обязательных соединений, или белков отображаются необычные переходы. Тем не менее, для многих лабораториях, эти подробный анализ слишком громоздки для повседневного использования. Поэтому мы предлагаем альтернативные методы лечения для различных сценариев, и продемонстрировать, как они могут быть использованы, чтобы соответствовать данные, вытекающие из различных белковых-лиганд. Наш метод использует инструмент StepOne КПЦР, для которых программное обеспечение для анализа на заказ данные доступны; пока это ускоряет анализ данных, результаты других инструментов могут быть обработаны с использованием ранее опубликованных методов 9, и то же самое после анализ может быть проведен.

<table><tbody><tr><td>StepOne real time PCR instrument</td><td>Life Technologies</td><td>4376357</td><td>DSF can be performed with many other instruments. The StepOne instrument has very convenient software for data analysis.</td></tr><tr><td>Protein thermal shift software v1.0</td><td>Life Technologies</td><td>4466037</td><td></td></tr><tr><td>MicroAmp Fast optical 48-well plates&amp;nbsp;</td><td>Life Technologies</td><td>4375816</td><td></td></tr><tr><td>Optical sealing tape&amp;nbsp;</td><td>Life Technologies</td><td>4375323</td><td>Bio-rad part no. 223-9444 is an alternative supplier</td></tr><tr><td>U-bottomed 96-well plates</td><td>Fisher</td><td>11521943</td><td></td></tr><tr><td>SYPRO Orange</td><td>Life Technologies</td><td>S6650</td><td>For a smaller volume supplier, use Sigma part no. S5692</td></tr><tr><td>SPSS statistics version 20</td><td>IBM</td><td></td><td>Other statistics packages will provide similar functionality</td></tr><tr><td>GraphPad Prism 6.02</td><td>GraphPad</td><td></td><td>Other statistics packages will provide similar functionality</td></tr><tr><td>Hand applicator (PA1)</td><td>3M</td><td>75-3454-4264-6</td><td></td></tr><tr><td>Hexokinase from Saccharomyces cerevisiae</td><td>Sigma-Aldrich</td><td>H5000</td><td></td></tr><tr><td>Glucose</td><td>Fisher scientific</td><td>10141520</td><td></td></tr></tbody></table>

determination of protein-ligand interactions using differential scanning fluorimetry

Широкий спектр методов в настоящее время доступны для определения константы диссоциации между белком и взаимодействующих небольших молекул. Тем не менее, большинство из них требуют доступа к специализированным оборудованием, и часто требуют степень экспертизы, чтобы эффективно создать надежные эксперименты и анализировать данные. Дифференциальная сканирующая флуориметрия (DSF) все чаще используется в качестве надежного способа первичного скрининга белков для взаимодействия малых молекул, либо для выявления физиологических партнеров или для открытия хит. Этот метод имеет то преимущество, что она требует только машину, подходящую для ПЦР количественной ПЦР, и таким образом, подходящие приборы доступна в большинстве учреждений; превосходный диапазон протоколов уже доступны; и есть все основания прецеденты в литературе для многократного использования метода. Прошлая работа предложил несколько способов расчета константы диссоциации из данных DSF, но это математически требовательный. Здесь мы деммонстрировать метод оценки константы диссоциации с умеренным количеством DSF экспериментальных данных. Эти данные, как правило, могут быть собраны и проанализированы в течение одного дня. Мы показываем, как разные модели могут быть использованы, чтобы соответствовать данные, полученные от простых обязательных мероприятий, и где кооперативного связывания или независимые сайты связывания присутствуют. Наконец, мы приведем пример анализа данных в случае, когда стандартные модели не применяются. Эти методы иллюстрируются по данным на коммерчески доступных белков управления, и двух белков из нашей исследовательской программы. В целом, наш метод обеспечивает простой способ для исследователей быстро получить дальнейшее понимание белка-лиганд с использованием DSF.

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Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

Определение белков-лиганд с использованием дифференциальной сканирующей флуориметрии

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

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61.5K Views.  University of Exeter. Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

Video: Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Анизотропию, как флуоресценция инструмента для изучения белок-белковых взаимодействий

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Biochemistry

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Дифференциальная сканирующая калориметрия - метод оценки термической устойчивости и конформации белка антигена

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Измерения биомолекулярных DSC профили с термолабильных лигандами быстро характеризовать складывания и привязки взаимодействия

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Фазового контраста и дифференциальных помех контраста (DIC) микроскопии

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Быстрое и количественный Флуориметрический Метод белка-таргетинга маленькая молекула наркотиками Скрининг

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Bioengineering

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Микрообъеме концентрации белка Определение использованием NanoDrop 2000c Спектрофотометр

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Нанодиафродная сканирующая флуориметрия для скрининга при обнаружении свинца на основе фрагментов

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Высокочувствительное детектирование связывания соединения с белком с помощью флуоресценции

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

В центре внимания автора: Оптимизация методов ПЦР для повышения доступности и эффективности

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data accuracy.

Детектирование взаимодействия белка CD40 с умбеллифероном с помощью дифференциальной сканирующей флуоресценции

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Определение белков-лиганд с использованием дифференциальной сканирующей флуориметрии

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