Laboratory Experiment pubs.acs.org/jchemeduc
Rapid and Adaptable Measurement of Protein Thermal Stability by Differential Scanning Fluorimetry: Updating a Common Biochemical Laboratory Experiment R. Jeremy Johnson,* Christopher J. Savas,† Zachary Kartje,§ and Geoffrey C. Hoops Department of Chemistry, Butler University, Indianapolis, Indiana 46208, United States S Supporting Information *
ABSTRACT: Measurement of protein denaturation and protein folding is a common laboratory technique used in undergraduate biochemistry laboratories. Differential scanning fluorimetry (DSF) provides a rapid, sensitive, and general method for measuring protein thermal stability in an undergraduate biochemistry laboratory. In this method, the thermal denaturation of multiple different proteins is determined in parallel using a reverse-transcription polymerase chain reaction (RT-PCR) machine and a hydrophobic dye that differentially binds to proteins in non-native conformations. The utility of this methodology is illustrated by the measurement of differential protein stability in microplate volumes, in triplicate, with small protein samples. These characteristics make DSF measurement of protein stability adaptable to use with noncommercial protein samples. The methodology is also expandable to quantitating protein stability under a wide variety of solution conditions. The rapid setup and analysis of DSF experiments not only provides advanced undergraduates with experience in a fundamental biochemical technique but also provides the adaptability for use in inquiry-based laboratories and in independent research projects. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Inquiry-Based/Discovery Learning, Biophysical Chemistry, Proteins/Peptides, Fluorescence Spectroscopy, Thermal Analysis, Undergraduate Research ince Anfinsen first proposed that the primary sequence of a protein determines its structure, uncovering the factors controlling protein folding have been a central focus of biomolecular research.1,2 Significant progress has been made in understanding the important variables controlling protein folding, even the de novo folding and design of proteins based on their amino acid sequence.2,3 Driving this expanded understanding of protein folding has been rapid advances in computer modeling and experimental techniques used to determine the structure and stability of proteins.4,5 One key technique used to assess the native stability of a protein is the measurement of thermodynamic parameters for protein unfolding, either by thermal or chaotropic denaturation.6,7 Protein denaturation follows the transition of the protein from its native (folded) conformation to a non-native (relatively unfolded) conformation. From the thermal denaturation curve, the midpoint of the transition (the Tm value) and the corresponding thermodynamic parameters for the protein’s folding can be determined.7 Protein denaturation experiments have been used to identify key residues involved in stabilizing native protein structure, to determine folding domains within proteins, and to calculate the contribution of specific intramolecular forces to controlling protein folding.8−11 The importance of protein folding and protein denaturation measurements to understanding protein function can also be inferred based on the number of different laboratory procedures published in this Journal for measuring protein
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© 2014 American Chemical Society and Division of Chemical Education, Inc.
stability.12−18 Laboratory experiments used to measure protein stability vary from traditional absorbance and relative enzymatic activity measurements to newer techniques using circular dichroism spectroscopy and Förster resonance energy transfer (FRET).12−18 Each of these measurements allows students to observe a transition in a protein’s structure and introduces students to the study of the conformational stability of proteins. However, current procedures require relatively large quantities of protein input, relegating thermal stability measurements to commercially available proteins whose thermal stability has already been well studied and validated. Current procedures also utilize instrumentation best suited for individual measurements, significantly reducing the throughput of the experiment. A new undergraduate laboratory experiment is described for measuring the thermal stability of proteins using differential scanning fluorimetry (DSF). The laboratory procedure for DSF requires only small volumes (10−25 μL) of a minimal protein concentration (0.100−0.300 mg/mL). A 96-well format allows the experiment to be simultaneously completed for an entire class in less than 2 h. These characteristics have made protein stability measurements using DSF compatible with an inquirybased, one-semester undergraduate biochemistry laboratory course. The goals of this experiment are for students to measure the folded stability of different protein variants and to Published: May 7, 2014 1077
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Figure 1. Representative thermal denaturation measured by DSF. (A) The temperature-dependent folded-to-unfolded transition for two bacterial hydrolases purified and characterized by biochemistry laboratory students (circles = Rv0045c; squares = ybfF). The unfolding of the protein and exposure of buried hydrophobic surface area lead to increased binding of the SYPRO Orange dye. The temperature at the midpoint of the unfolding transition is defined as the Tm value and can be used to compare the thermal stability of different proteins. (B) The first derivative of the thermal denaturation curve provides a clear marker of the Tm value for the two proteins. Each thermal denaturation was completed in triplicate and is displayed with their standard error. The error bars for many values are not visible because the error was smaller than the size of the data marker.
dilutions between 1:100 and 1:1000, allowing one commercial sample of fluorogenic dye to be used in many DSF experiments.26,27 The ease of setting up DSF experiments and their scalability to high-throughput, automated instrumentation has made them common secondary screening assays in the pharmaceutical industry.20,21 Widespread adoption of DSF has coincided with the growth in availability of RT-PCR machines and the introduction of modules for measuring protein thermal stability into standard RT-PCR software. Many different RT-PCR machines from across many vendors are now compatible with DSF measurements, allowing the majority of colleges and universities to now have access to DSF.21
assess the role of specific amino acids in stabilizing the native conformation of a protein.
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BACKGROUND ON DSF TECHNIQUE DSF (also known as Thermofluor) takes advantage of the sensitivity, high thermal control, and expanded availability of reverse-transcription polymerase chain reaction (RT-PCR) machines to measure protein thermal stability.19−21 DSF analysis has been applied to a variety of biochemical problems including determination of relative thermal stabilities between protein variants, optimization of stabilizers for protein crystallography, and even high-throughput screening for small molecule inhibitors.19,22,23 The last two applications exploit the increased thermal stability of proteins when bound to stabilizing ligands or in a stabilizing solution to measure their relativity affinity.19 The shift in the Tm value between the inhibitor-bound and unbound state provides a measure of the affinity between the inhibitor and the protein and can be used to derive the thermodynamics for the interaction.24,25 In a basic DSF experiment, the protein of interest and an environmentally sensitive fluorogenic dye are incubated together in a microplate and the relative fluorescence measured at increasing temperatures.19−21 The fluorogenic dyes used in DSF experiments have fluorescence dependence on their local hydrophobic environment. Thus, as an aqueous protein unfolds and exposes the hydrophobic core of the protein, more fluorogenic dye binds, increasing the fluorescence. By fitting the relative fluorescence versus the temperature, the Tm value can be determined.19,23 Because the change in fluorescence is dependent on the binding of fluorogenic dye, specific fluorogenic dyes are required to obtain reliable DSF measurements. The most common fluorescent dye used for DSF measurements is SYPRO Orange.21 SYPRO Orange has a fluorescence minimum and maximum that are well matched with the common filters provided on RT-PCR machines (excitation ∼470 nm and emission at ∼570 nm) and a proportionally large increase in fluorescence upon binding to hydrophobic proteins.21 Unfortunately, the chemical structure of SYPRO Orange is proprietary, removing the ability to have discussions of the structure−function relationship between dye binding and fluorescence. Alternative fluorogenic dyes have also been used, including anilinonaphthalenesulfonic acid (ANS) and SYPRO Ruby.21,26 Although the dyes used to measure stability are not inexpensive, the dyes are typically used at
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EXPERIMENTAL PROCEDURE Student-purified proteins (either ybfF from Vibrio cholerae or Rv0045c from Mycobacterium tuberculosis) were diluted to a final concentration of 0.300 mg/mL in phosphate buffered saline.27−29 Diluted protein samples were then added in triplicate to a PCR microplate. A small aliquot of a 1:20 dilution of SYPRO Orange protein stain was added to give a final dilution of SYPRO dye of 1:500. As the absolute concentration of the dye was not provided by the commercial vendor, the only method for measuring the final concentration of SYPRO Orange dye was by relative dilution scales. The protein samples sealed in the PCR plate were then heated from low-to-high temperature (15 to 80 °C) at a slow rate (1−1.5 °C/min) in an RT-PCR machine. The change in SYPRO Orange fluorescence was monitored over time (λex = 450−490 nm, λem = 610−650 nm). For analysis, a two-state model was assumed where unfolding of the native conformation to a non-native conformation caused greater fluorescent dye binding. The denaturation midpoint between these two states (Tm) was determined by plotting the first derivative of fluorescence versus temperature and finding the temperature at the midpoint of the transition. Students utilized the data analysis software provided with the instrument to correct for background drift and to determine the Tm values for their proteins. Graphs are usually presented as normalized plots where the average minimum fluorescence is set to 0 and average maximum fluorescence is set to 1.19,26,27 A detailed version of the experimental procedure is provided in the Supporting Information. 1078
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HAZARDS
The relative toxicity of SYPRO Orange is not known, but suggested precautions include wearing double gloves when handling the dye as it is dissolved in dimethyl sulfoxide. Dimethyl sulfoxide can be readily absorbed through the skin and is a potential irritant. Used dye and protein denaturation PCR plates should also be disposed of using proper Unwanted Material disposal.
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RESULTS AND DISCUSSION In a one-semester biochemistry laboratory course, students purified unique variants of a chosen bacterial hydrolase and then determined the biochemical characteristics of their protein variants. One biochemical characteristic that students measured was the thermal stability of their protein variants in comparison to the wild-type enzyme. Using DSF for measuring protein thermal stability allowed an entire class of 8−14 students to measure all thermal denaturation curves in triplicate within 2 h, thus simultaneously measuring the thermal stability of all protein variants. Representative student data of the thermal denaturation curves for two wild-type hydrolases are shown in Figure 1. The two hydrolases shown are ybfF from Vibrio cholerae and Rv0045c from Mycobacterium tuberculosis. These two hydrolases are structural and sequence homologues that have been used as good model systems for understanding protein structure− function relationships.27−29 The relative increase in the fraction unfolded with increasing temperature corresponded to an increase in SYPRO Orange dye binding. Maximal fluorescence and dye binding was reached when the protein was completely in the unfolded state. 19,23 The observed decrease in fluorescence after reaching the maximal fluorescence value for some proteins (see Rv0045c in Figure 1A) was a common artifact measured in DSF.19,23 This effect was attributed to protein aggregation at higher temperatures, which reduced the surface area for dye binding, and thus decreased the overall fluorescence.19 The analysis software for the RT-PCR machine corrected for some of this drift when presenting the first derivative of the dye fluorescence (Figure 1B). The first derivative plot provided a clear, visual representation of the Tm value for each protein and helped with comparison of relative Tm values between wild-type proteins and their protein variants (Figure 2). For instance, two studentproduced protein variants of the ybfF hydrolase (F158A and H235A) showed significant decreases in their Tm values (ΔTm = 3 and 4 °C, respectively) relative to the wild-type protein. These decreases were based on the hydrophobicity of the amino acid substituted and their relative burial within the protein’s native conformational structure.27 For the two hydrolases, differences in thermal stabilities were measured between 1 and 10 °C, although greater differences were observed for proteins with higher thermal stabilities than the two hydrolases studied here.26 Students prepared formal lab reports comparing the folded stability of their protein variants to the wild-type enzyme. In these reports, students were assessed based on their ability to analyze their experimental data and to propose molecular explanations for the differences in the stability of their protein variants. DSF analysis of student protein variants has been run reproducibly for four laboratory cycles with over 25 protein variants and 120 different student protein samples. To ensure reproducible DSF results, samples should be loaded into the
Figure 2. Thermal stability of student protein variants. Relative thermal stability of a wild-type hydrolase (ybfF) in comparison to two different student protein variants. The first derivative of the unfolding curve is shown to highlight the decrease in Tm values with each substitution. Each thermal denaturation was completed in triplicate and is displayed with their standard error.
center lanes on the plate, as significant evaporation has been observed from the outside lanes. However, this may differ based on the RT-PCR machine and plate seal used. The relative rate of sample heating is also an important variable to consider, as the heating rate can affect the relative unfolding of a protein.30 Within the given range (1−1.5 °C/min), no effects of the heating rate on the Tm value have been observed. Finally, although a two-state model matches the unfolding curves for these bacterial hydrolases, a two-state model cannot explain all protein unfolding pathways. Models for fitting DSF data with more complex protein unfolding pathways are available and could be adapted for biophysical chemistry courses studying protein folding.20,31
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CONCLUSIONS Overall, DSF provided the versatility, sensitivity, and speed necessary to be used as a general method for measuring protein thermal stability in a variety of laboratories. The increased accessibility of RT-PCR machines and the adaptability of DSF allowed standard protein stability measurements to be redesigned for an inquiry-based biochemistry laboratory. DSF measurements were also adaptable to a variety of student classroom projects or expandable into further experiments on protein folding as described in the Supporting Information.
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ASSOCIATED CONTENT
S Supporting Information *
Instructor notes with possible additional experiments and student handout. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*R. J. Johnson. E-mail:
[email protected]. Present Addresses †
University of IllinoisChicago Dental School, Chicago, Illinois 60612, United States. § Department of Chemistry and Biochemistry, Southern Illinois UniversityCarbondale, Carbondale, Illinois 62901, United States. 1079
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Notes
(19) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2007, 2 (9), 2212−2221. (20) Senisterra, G. A.; Finerty, P. J., Jr. High throughput methods of assessing protein stability and aggregation. Mol. BioSyst. 2009, 5 (3), 217−223. (21) Simeonov, A. Recent developments in the use of differential scanning fluorometry in protein and small molecule discovery and characterization. Expert Opin. Drug Discovery 2013, 8 (9), 1071−1082. (22) Lavinder, J. J.; Hari, S. B.; Sullivan, B. J.; Magliery, T. J. Highthroughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J. Am. Chem. Soc. 2009, 131 (11), 3794−3795. (23) Boivin, S.; Kozak, S.; Meijers, R. Optimization of protein purification and characterization using Thermofluor screens. Protein Expr. Purif. 2013, 91 (2), 192−206. (24) Holdgate, G. A. Thermodynamics of binding interactions in the rational drug design process. Expert Opin. Drug Discovery 2007, 2 (8), 1103−1114. (25) Zhang, R.; Monsma, F. Fluorescence-based thermal shift assays. Curr. Opin. Drug Discovery Devel. 2010, 13 (4), 389−402. (26) Hedge, M. K.; Gehring, A. M.; Adkins, C. T.; Weston, L. A.; Lavis, L. D.; Johnson, R. J. The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima. Biochim. Biophys. Acta 2012, 1824 (9), 1024−1030. (27) Ellis, E. E.; Adkins, C. T.; Galovska, N. M.; Lavis, L. D.; Johnson, R. J. Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase. ChemBioChem 2013, 14 (9), 1134− 1144. (28) Guo, J.; Zheng, X.; Xu, L.; Liu, Z.; Xu, K.; Li, S.; Wen, T.; Liu, S.; Pang, H. Characterization of a novel esterase Rv0045c from Mycobacterium tuberculosis. PLoS One 2010, 5 (10), e13143. (29) Zheng, X.; Guo, J.; Xu, L.; Li, H.; Zhang, D.; Zhang, K.; Sun, F.; Wen, T.; Liu, S.; Pang, H. Crystal structure of a novel esterase Rv0045c from Mycobacterium tuberculosis. PLoS One 2011, 6 (5), e20506. (30) Senisterra, G.; Chau, I.; Vedadi, M. Thermal denaturation assays in chemical biology. Assay Drug Dev. Technol. 2012, 10 (2), 128−136. (31) Senisterra, G. A.; Soo Hong, B.; Park, H. W.; Vedadi, M. Application of high-throughput isothermal denaturation to assess protein stability and screen for ligands. J. Biomol. Screening 2008, 13 (5), 337−342.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS R.J.J. and G.C.H. gratefully acknowledge support for the laboratory from a National Science Foundation grant (DUE1140526). We are grateful to the students from CH463 20102013 for piloting the thermal stability measurements. We are grateful to Hai Pang (Tsinghua University) who kindly provided the expression plasmid for Rv0045c.28,29
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