nanodsf Precisely revealing protein folding and stability 2bind: Your service provider for biophysical characterization of proteins
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Purpose Thermodynamic stability is fundamental for the biological function of proteins. Information on protein stability is, therefore, essential for the study of protein structure and folding and can also be used to indirectly monitor protein-ligand interactions. This booklet describes the innovative nanodsf technology, which is used to determine protein folding and stability. Overview Applications Technology Determining protein stability Example measurement Experimental consideration Comparing stability assay methods
Fast protein characterization Measuring protein folding and stability within minutes nanodsf assays by 2bind nanodsf, a very sensitive and fast method to analyze and characterize protein folding and stability. The nanodsf approach utilizes the changes in the intrinsic tryptophan or tyrosine fluorescence that occur upon protein unfolding. Thermal unfolding of protein samples is analyzed in a broad concentration range on a temperature gradient within minutes. Up to 48 different samples (e.g. 48 buffer conditions for one sample) in a single run.
Overview nanodsf is a novel label-free method used for thermal and chemical unfolding experiments to monitor and quantify structural stability of proteins. Intrinsic tryptophan fluorescence of proteins is strongly dependent on the 3D-structure, hence the surrounding of the amino acid (see figure to the right). Using chemical denaturants or a thermal gradient, protein structures are unfolded, leading to changes in fluorescence intensity and / or in the wavelength of the emission maximum. nanodsf monitors these fluorescence changes with high resolution and is even capable of revealing multiple unfolding transition points. Therefore, nanodsf is especially useful in antibody engineering, membrane protein characterization, formulation development and protein quality control. F330/350 ratio 1.2 1 0.8 0.6 0.4 0.2 0 folded unfolded Temperature (in C) or Conc. (denaturant) Principle behind the nanodsf. Increasing temperature causes protein unfolding that can be assessed by monitoring changes of tryptophan fluorenscence at 330nm and 350nm wavelength.
Looking deep into details A method with ultra-high resolution nanodsf allows to identify any unfolding transition state nanodsf is able to measure the stability of proteins without any labelling with ultra-high resolution. The combination of up to 36000 data points per measurement and the broad temperature range from 15-95 C permits the precise determination of even multiple unfolding transition states.
Key Advantages of nanodsf molecular by interactions 2bind Applications nanodsf is the method of choice for protein stability analyses. nanodsf offers an easy, rapid and accurate way to decipher changes in protein folding states that contribute to a better engineering of antibodies, galenics and others. In contrast to conventional DSF methods, nanodsf measurements are performed with unmodified proteins close to their native state. This allows analysis of proteins and protein assemblies that were previously problematic to investigate. Furthermore, using nanodsf, molecules can be assessed in a broad concentration range and independent of their size and buffer conditions. The whole spectrum, from small peptides to protein complexes, can be studied with an unique high sensitivity. nanodsf assays performed at 2bind: 1. Stability screening assays: optimization of formulation conditions (also viscous solutions) buffer screening assays to identify the optimal conditions detergent screening assay to determine the optimal conditions for membrane proteins 2. Biophysical characterization assays: antibody + antibody-drug conjugate characterization determination of multiple domain unfolding transitions 3. Quality control assays: long term stability of proteins forced degradation of proteins 4. Ligand binding screening assays
Key advantages of nanodsf assays performed by 2bind no dye required only 10 µl sample required simultaneous measurement of 330nm and 350nm for optimal data quality from 15 C to 95 C up to 36000 data points per measurement free choice of buffers and detergents 48 capillaries scanned within minutes broad concentration (5 µg/ml to 200 mg/ml) and size range
Label-free analyses Detection of intrinsic tryptophan fluorescence nanodsf reveals protein folding and stability in a label-free manner Any molecule that possesses tryptophans (or tyrosines) in its sequence can be measured in nanodsf assays by the intrinsic fluorescence of these amino acids. In contrast to conventional DSF assays, no fluorescent dye has to be attached, thus avoiding undesirable stabilisation of the unfolded protein state by the dye. In addition, there is no limitation in the use of detergents, which might interact non-specifically with the dye. nanodsf is the best method for close to native state analyses of protein structures.
nanodsf technology nanodsf is a differential scanning fluorimetry method that assesses the stability of molecules through a temperature gradient or chemical denaturation. Generally, the thermal stability of a protein is described by the thermal unfolding transition midpoint Tm ( C), the point at which half of the proteins are unfolded. The nanodsf measurement is performed in a label-free manner based on the measurement of the intrinsic tryptophan fluorescence of proteins. This fluorescence relies on the close surrounding of amino acids and changes upon thermal unfolding. The fluorescence wavelength ratio of 330-350 nm (described in the literature as the tryptophan fluorescence emission shift) upon protein unfolding is used by nanodsf to calculate Tm of single and multiple transition states. The technical setup of the nanodsf device (Prometheus NT.48, NanoTemper Technologies, Munich, Germany) is shown in the picture to the right. Technical setup of nanodsf. The aqueous protein solution is heated in a small area of a capillary with a maximal thermal gradient of 15-95 C. Proteins are excited by a LED laser and changes of intrinsic trytophan fluorescence emission is then detected in 48 different conditions within minutes. Picture provided by NanoTemper
molecular interactions Low sample consumption Free choice of buffer conditions Protein stability analysis at low sample consumption and free choice of buffers The nanodsf technology allows for characterization of protein stability in 48 different experimental conditions in just one run with free choice of buffers and detergents. Only small amounts of protein sample (10 µl) are needed for an accurate analysis. Concentrations from 5 µg/ml to 200 mg/ml can be analyzed in this thermal shift assay.
Calculating the thermal unfolding transition midpoint (Tm) folded 4000 330nm 350nm Fluorescence 4 2 transition Fluorescence 3000 2000 unfolded 0 300 320 340 360 380 400 420 Wavelength, nm 1000 0 10 20 30 40 50 60 70 80 90 Temperature, C Dual UV-detection system of the nanodsf. The folded state of a protein presents a higher tryptophan fluorescence intensity than the unfolded state (left). Intrinsic tryptophan fluorescence is measured at 330nm and 350nm wavelength and plotted against the temperature from 15-95 C (right). Tryptophan is a common hydrophobic amino acid, mostly located in the hydrophobic core of proteins shielded from the aqueous solvent. Upon unfolding, tryptophan is exposed and changes, firstly, its fluorescence intensity, and secondly, its emission peak (blue or red shift). nanodsf precisely detects the change of intrinsic tryptophan fluorescence with its dual UV-detection system at 330nm and 350nm wavelength as demonstrated in the image on the left. Recording fluorescence at 330nm and 350nm allows the measurement of even minor differences in fluorescence intensity and fluorescence emission peaks, which are undetectable in a single wavelength measurement as indicated in the image on the right.
Calculating the thermal unfolding transition midpoint (Tm) 1.2 F330/ 350 ratio By plotting the fluorescence ratio of F330/350 against the temperature, even tiny differences become detectable (upper pannel). Furthermore, by analyzing the ratio, the influence of fluorescence background (e.g. by compounds or co-factors) is negligible. After plotting the fluorescence ratio against the temperature, the melting temperature Tm is determined by first derivate analysis, which accurately describes the thermal unfolding transition midpoint (lower pannel). Fluorescence 1.1 1.0 0.9 0.8 0.7 10 20 30 40 50 60 70 80 90 Temperature, C 79.1 C The protein given in the example possesses one unfolding transition point and the Tm is determined to be 79.1 C. 10 20 30 40 50 60 70 80 90 Temperature, C Example Tm measurement. Upper panel, F330/350 fluorescence ratio intensity of intrinsic tryptophan plotted against temperature. Lower pannel, Tm calculation by first derivate analyses.
Example analysis: Buffer optimization for ERK2 (MAPK1) nanodsf is a rapid and precise technology to study thermal stability of proteins under different buffer conditions. ERK2 (MAPK1, mitogen-activated protein kinase 1) is an important player in many signalling pathways associated with cancer progression. In this example, we analyzed the thermal stability of ERK2 under 28 different buffer conditions. Tm was calculated in different buffers with different salt concentrations in a broad ph range (4.7 to 8.0). Results of these assays present ERK2 reaction buffer to be the most adequate buffer for a high ERK2 stability with a Tm of 54.9 C. After boiling the protein for 5 min in citric acid ph 3.5 the protein is completely unfolded, no Tm was identified, which serves as a negative control. Buffer optimization assay of ERK2. Comparision of 28 different buffer conditions. *ERK2 reaction buffer containing 12.5 mm b-glycerophosphate, 7.5 mm MgCl 2, 0.5 mm NaF, 0.5 mm vanadate
Experimental consideration To ensure optimal nanodsf results, different technical aspects have to be considered in the experimental setup. Fluorescence nanodsf works in a label-free fashion measuring intrinsic fluorescence of tryptophan and tyrosine within proteins. Usually, one tryptophan in the protein is enough to proceed with an accurate nanodsf assay. Buffers nanodsf offers free choice of buffers. There are no restrictions to buffer substances or salt concentrations. nanodsf is the optimal tool to determine the buffer conditions providing optimal thermal stability. Temperature range nanodsf analyses are usually performed in a temperature gradient of 15-95 C, with a heating rate of 1 C per min. However, these settings can be adapted for the specific protein. Capillaries Depending on the fluorescence intensity, two different types of capillaries can be chosen for nanodsf assays to ensure optimal signal to noise ratios. Detergents nanodsf assays can be performed using any kind of detergent. This is of special interest for membrane protein characterization. Assay quality controls To ensure highest data quality, it is essential to run positive and negative controls with the samples of interest. The controls ensure that the technical setup is optimal and help to interprete the final data.
Appendix What is the best way to characterize protein stability? Comparing nanodsf with orthogonal methods Different methods to study protein stability rely on different principles thus pose different requirements towards substance concentration, buffer compositions and setup labor. nanodsf performed by 2bind is time- and cost-efficient hence the best choice in almost every aspect.
Facts and Features Method Advantages Drawbacks 2bind nanodsf - protein stability analysis under close to native conditions - low sample consumption - free choice of buffers and detergents - ultra-high resolution due to high data point density - rapid analysis - unfolding transitions of proteins lacking tryptophans might be difficult to detect Differential Scanning Fluorimetry (DSF) - measurement can be done by a conventional q-pcr machine - dyes may affect structural protein unfolding transitions by stabilization of the unfolded state - probability of background noise due to detergent dye complexes - not applicable to all biomolecular species Differential Scanning Calorimetry (DSC) - direct measurement of thermodynamic properties - analysis of biomolecules to nano-sized materials - large amount of sample is necessary - analysis is sensitive to aggregation, precipitation and self-association effects Circular Dichroism Spectroscopy (CD) - additional information on protein configuration, (distribution of alpha-helix or beta-sheet, etc.) - requirement of specific buffer conditions - detergents absorb light and affect measurement
2bind take home message nanodsf protein stability assays
nanodsf assays by 2bind Be clever - Work with the experts 2bind: The provider for nanodsf services Dr. Thomas Schubert with Prof. Gernot Längst (University of Regensburg)
2bind GmbH Josef Engert Str. 13 D 93053 Regensburg Handelsregister B Regensburg: HRB12909 info@2bind.de www.2bind.de Dr. Thomas Schubert schubert@2bind.de +49 160 96938061