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Thermal unfolding Application Note NT-PR-001

Analyzing Thermal Unfolding of Proteins: The Prometheus NT.48 Lea Martin, Sascha Schwarz & Dennis Breitsprecher

NanoTemper Technologies GmbH Floessergasse 4 81369 Munich, Germany

Abstract

A detailed analysis of protein stability is a prerequisite for both, the basic understanding of protein folding mechanisms as well as for the successful development of biologicals in the pharmaceutical industry. Here we demonstrate the performance of the new Prometheus NT.48 instrument, which detects intrinsic protein fluorescence changes upon thermal or chemical unfolding of up to 48 samples in parallel.

Introduction

The assessment of protein thermal stability is an integral part in basic research, drug discovery and drug development [1]. For instance, shifts in the melting temperature (Tm) of a target protein upon binding to a small molecule ligand are routinely used in primary screens in the drug discovery process [2]. In addition, the thermal and chemical stability of biologicals, such as antibodies, is often monitored to establish optimal conditions for large-scale production and long-term storage [3, 4]. Moreover, the careful analysis of protein unfolding and refolding mechanisms can yield important insights into the thermodynamic origins of protein folding, thereby helping to elucidate the molecular basis of degenerative diseases such as Alzheimer, Parkinson or diabetes [5]. The basis of label-free fluorimetric analysis of protein folding lies in the properties of the fluorescent amino acid tryptophan. Since tryptophan is a hydrophobic amino acid, it is mostly located in the hydrophobic core of proteins where it is shielded from the surrounding aqueous solvent. Upon unfolding however, tryptophan is exposed, which alters its photophysical properties [6]. By detecting changes in tryptophan fluorescence intensity and its emission peak shift, the transition of a protein from the folded to the

unfolded state can be precisely recapitulated. This way, the melting temperature (Tm) and thermodynamic parameters can be determined [7]. Here we demonstrate the performance of the Prometheus NT.48 in monitoring thermal unfolding of proteins in a formulation screening project. The Prometheus NT.48 can measure up to 48 samples in parallel, and uses high-precision capillaries which are filled with just 10 µl of sample. Using a detector which is specifically designed to monitor changes in the emission spectrum of tryptophan with maximal sensitivity and speed, highest data point density and precision is achieved.

Figure 1 Structure of pig pancreatic -amylase (PPA, green)

and Aspergillus oryzae -amylase (TAKA, blue). The red sphere represents a Ca2+-ion.

Proteins of the -amylase family are well-established for the analysis of protein folding [8]. Most amylases share very similar tertiary

structures, with three ()-barrel domains and at least one conserved Ca2+-binding site (Figure 1). At the same time however, they show an extremely broad range of melting temperatures

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(from 40 °C to 110 °C), which made them perfect candidates for basic research on the determinants of thermal stability of proteins [9]. In addition to their value for basic medical research, amylases are commercially used in the production of ethanol in sugars in large-scale industries. In the present example, we investigated the

thermal unfolding of -amylase from mammals

(Pig pancreatic -amylase, PPA) and fungi

(Aspergillus oryzae -amylase, TAKA). We recapitulated the stabilizing effects of Calcium ions on protein conformation, and finally performed formulation screens using different additives which improve thermal stability to different degrees.

Results

Monitoring and Analyzing Thermal Unfolding using the Prometheus NT.48

The Prometheus NT.48 monitors the shift of intrinsic tryptophan fluorescence of proteins upon unfolding by detecting the fluorescence at an emission wavelength of 330 and 350 nm. For determination of the protein melting point (Tm, where half of the protein is folded and the other half is unfolded), either the fluorescence change in one of the two channels can be used, or alternatively, the ratio of the fluorescence intensities (F350/F330) can be plotted.

Figure 2 Analysis of TAKA and PPA melting curves. (A) Plot of the tryptophan fluorescence decay upon thermal unfolding of TAKA (left). The transition from the folded to the unfolded state is already visible in the raw fluorescence data at emission wavelengths of 330 and 350 nm. The inset demonstrates the high data point density of the Prometheus NT.48. To determine Tm, the experimental data can be fitted with a polynomial function. Its first derivative displays a peak at the point of maximal slope, which corresponds to Tm (right). (B) Equivalent analysis of Tm for PPA. Note that in contrast to TAKA, the transition of folded to unfolded protein is not visible in the raw fluorescence data (left), while Tm can be easily determined from the fluorescence ratio plots (right).

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The latter approach is preferred for most proteins, since the fluorescence ratio monitors both, the change in tryptophan fluorescence intensity as well as a shift of the fluorescence emission maximum towards higher wavelengths (“redshift”) or lower wavelengths (“blueshift”). Thermal unfolding of PPA and TAKA was performed at a heating rate of 1 °C/minute, resulting in a data point density of 10 points/°C, which allows for a precise determination of the onset of protein unfolding as well as for precise fitting of the folded-unfolded transition by mathematical models. Figure 2 shows the changes of tryptophan fluorescence of PPA and TAKA upon thermal unfolding. Notably, for TAKA, the raw fluorescence data from both wavelengths show a clear transition from folded to unfolded (Figure 2A, left) which could be directly used for Tm analysis. In contrast, this transition is not evident from the raw data for PPA (Figure 2B, left). Moreover, while TAKA displayed a typical unfolding profile with a shift of the tryptophan fluorescence towards higher wavelengths (redshift), PPA showed a less common shift of the tryptophan fluorescence towards lower wavelengths (blueshift).

A plot of the fluorescence ratio F350/F330 of both proteins against the temperature yielded clear melting curves which could be used for analyzing the respective melting temperature of the amylase isoforms. Determination of the melting temperature can be performed using different methods. A commonly used approach is to determine the maximum of the first derivative of the absorbance signal. This method circumvents the somewhat subjective determination of baseline levels (Figure 2A and B, right), and also allows for the determination of multiple melting points, e.g. for antibody unfolding or more complex multi-domain proteins. Most importantly, the standard deviation of the results shown in the table in Figure 3B from the first derivative analysis is in the range of the error of the fit, demonstrating the maximal reproducibility of the results. Thus, the Prometheus NT.48 can be used to precisely determine Tm values with a minimum of sample and time consumption. The results show that both, reproducibility and accuracy of thermal unfolding experiments with PPA and TAKA were very high (Figure 3A and B). The obtained Tm values closely matched reported values from the literature [9].

Figure 3 Precision and reproducibility of Prometheus NT.48 unfolding data. (A) The plots represent an overlay of 10 independently recorded melting curves of PPA and TAKA, respectively. (B) Determination of Tm for both proteins displays a small standard deviation between experiments (≤ 0.2 °C) and a good correlation with published results [9].

Ca2+-Dependence of Amylase Thermal Stability

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In a second set of experiments, we aimed to recapitulate the stabilizing effects of Ca2+-Ions on

both -amylase isoforms. Ca2+-Ions have previously been shown to be required for increased Tms of different amylase isoforms, ranging from virtually no effect for Alteromonas amylase to an increase of Tm by 50 °C for Bacillus Licheniformis amylase [9]. In order to study the effects of Ca2+-ions on PPA and TAKA stability, we incubated both proteins in

buffer with 5 mM EDTA to remove bound Ca2+ for 30 minutes prior to thermal unfolding experiments. As expected, removal of Ca2+-ions by EDTA resulted in a marked decrease in Tm for both

amylase isoforms (Figure 4). Tm was more pronounced for PPA (-16.6 °C) than it was for TAKA (-12 °C), which correlates nicely with previously published results (PPA -17 °C, TAKA -14 °C) [10, 11].

Figure 4 Ca2+ effects on amylase stability. Removal of Ca2+ ions results in a marked destabilization of both amylase isoforms, as indicated by the shift of Tm towards lower values.

Effects of buffer additives on amylase thermal stability.

A screening for additives and buffer conditions that improve protein stability, also referred to as formulation screening, is of key importance to achieve maximal shelf life of antibodies and other biologicals. Using the Prometheus NT.48, we tested the effects of different buffer additives that were previously shown to increase protein stability, namely glycerol, sucrose, trehalose and sorbitol, at concentrations ranging from 10 % to 40 % (w/v), on PPA and TAKA. The formulation screen of 16 different buffer conditions for each amylase isoform was performed in a single run, with a temperature range from 20 °C to 90 °C and a heating rate of 1 °C/min. Measurements were performed within

~ 70 minutes, with a total sample consumption of 400 µl (10 µl for each buffer condition + 4 control experiments for each isoform without additive), and a total amount of protein of just 80 µg. The plots of the tryptophan fluorescence ratios clearly show that each additive increased Tm of PPA and TAKA in a concentration dependent manner. For PPA, trehalose was most effective already at concentrations of 30 % (+ 12 °C), while glycerol was least effective, increasing Tm by just 7.5 °C at a concentration of 40% (Figure 5A and B). For TAKA, addition of 40 % sucrose proved to be most effective in increasing Tm (+ 12 °C), while glycerol and trehalose showed the smallest effect (+ 7.5 °C and + 8 °C, respectively) (Figure 6A and B). These results are in good agreement with a previous study investigating the effect of additives

on Bacillus -amylase thermal unfolding [12].

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Figure 5 Formulation screening of PPA. In order to find optimal conditions for an increased thermal stability of PPA, its thermal unfolding was monitored under 16 different additive conditions. A clear shift of Tm towards higher values is visible in the fluorescence ratio plots for each additive. Quantification of Tm under different conditions renders the addition of 30 % trehalose most effective, while glycerol has the weakest effect.

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Figure 6 Formulation screening of TAKA. In order to find optimal conditions for an increased thermal stability of TAKA, its thermal unfolding was monitored under 16 different additive conditions. A clear shift of Tm towards higher values is visible in the fluorescence ratio plots for each additive. Quantification of Tm under different conditions renders the addition of 40 % sucrose most effective, while glycerol and trehalose have the weakest effect.

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Conclusions In this case study, we demonstrate the performance of the NanoTemper Technologies instrument Prometheus NT.48 in determining

thermal unfolding properties of two -amylase isoforms in screening approaches.

By detecting changes in tryptophan fluorescence at two defined wavelengths, Tm values of the amylase proteins could be determined under different conditions. All results show a good agreement with published values. Most notably however, when compared to approaches using standard fluorimeters, both sample consumption and the time required to perform the experiments are dramatically reduced by using the Prometheus NT.48.

The capillary format of the instrument allows for a flexible experiment design, measuring any number of samples between 1 and 48 simultaneously. Importantly, the use of Prometheus capillaries offers an even higher precision for UV-fluorescence detection than high-performance quartz-cuvettes, with the additional benefit of low sample consumption, high-throughput and high versatility. Moreover, the capillary-based approach prevents cross-contamination, and no laborious and time-consuming cleaning steps are required. High scanning rates and thus high data density moreover allow for a robust analysis of melting curves by mathematical fitting algorithms, and also enables a precise determination of unfolding onsets.

In addition, the direct detection of tryptophan fluorescence to monitor protein unfolding has several benefits compared to other methods routinely used to monitor thermal unfolding, such as differential scanning fluorimetry (DSF) or thermofluor assays. These assays use external fluorophores which bind to hydrophobic patches of the protein usually buried in the core of the protein. Upon unfolding, these patches get exposed and the fluorophore attaches resulting in an increase in fluorescence. These assays, however, are not suited for a detailed analysis of folding thermodynamics, since they interfere with folding-unfolding equilibria by directly interacting with the proteins. Moreover, the external fluorophores are incompatible with a number of buffers (e.g. including detergents) or protein types, such as membrane proteins. Lastly, although DSF is routinely used in primary screenings in the drug discovery process, external fluorophores can interact with compounds or block binding sites and produce false-negative as well as false-positive results.

In addition to its capabilities in monitoring thermal unfolding of a large number of samples in parallel,

the Prometheus NT.48 can also be used to analyze chemical denaturation of proteins within seconds. In summary, our results demonstrate that the Prometheus NT.48 is exceptionally well suited for a rapid, precise and cost-effective characterization of protein stability, both in academic and industrial settings. Its flexibility and speed make it a valuable tool for a plethora of different experimental approaches, ranging from in-depth characterization of protein folding to high-throughput screening projects.

Material and Methods

Sample preparation α-amylase from pig (pig pancreatic α-amylase, PPA, Roche) and α-amylase Aspergillus oryzae, TAKA, Sigma) were dissolved in 30 mM HEPES, 50 mM NaCl, 2 mM CaCl2, pH 7.4 at concentrations of 10 mg/ml. Final concentrations in thermal unfolding experiments were 10 µM. In order to remove residual traces of ammonium sulfate or other contaminants, a buffer exchange using buffer exchange spin columns was performed (NanoTemper Technologies). For the determination of the Ca2+-dependence of α-amylase stability, a second buffer exchange into buffer lacking CaCl2 but including 5 mM EDTA was performed. For the formulation screen, the proteins were transferred into 20 mM Na-Citrate buffer, pH 5.9, with the respective concentrations of sucrose, sorbitol, trehalose or glycerol.

Thermal unfolding experiments For thermal unfolding experiments, the proteins were diluted to a final concentration of 10 µM. For each condition, 10 µl of sample per capillary were prepared. The samples were loaded into UV capillaries (NanoTemper Technologies) and experiments were carried out using the Prometheus NT.48 . The temperature gradient was set to an increase of 1 °C/min in a range from 20 °C to 90 °C. Protein unfolding was measured by detecting the temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm. Data Analysis Melting temperatures were determined by detecting the maximum of the first derivative of the fluorescence ratios (F350/F330). For this, a 8th order polynomial fit was calculated for the transition region. Next, the first derivative of the fit was formed and the peak position (at Tm) was determined.

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