Development and Implementation of a Protein ... - ACS Publications

Laboratory Experiment pubs.acs.org/jchemeduc

Development and Implementation of a Protein−Protein Binding Experiment To Teach Intermolecular Interactions in High School or Undergraduate Classrooms Sadie M. Johnson, Cassidy Javner, and Benjamin J. Hackel* Chemical Engineering and Materials Science, University of Minnesota−Twin Cities, 421 Washington Avenue Southeast, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: The goal of this study was to create an accessible, inexpensive, and engaging experiment to teach high school and undergraduate chemistry or biology students about intermolecular forces and how they contribute to the behavior of biomolecules. We developed an enzyme-linked immunosorbent assay (ELISA) to probe specific structure−function relationships in the context of a protein−protein interaction that can be completed within a week of 45 min daily classes or a single 3−4 h lab using accessible reagents and materials (e.g., micropipettes and camera phones). The assay detected the high-affinity interaction between immunoglobulin G (IgG) and an engineered fibronectin domain protein. To demonstrate the impact of small chemical changes on intermolecular interactions, four mutant fibronectin domains were engineered, each with a single amino acid change, to provide a variety of chemical groups in the hypothesized binding site that resulted in a range of affinities for IgG (equilibrium dissociation constants from 1.5−696 nM). The experiment was implemented with two classes of high school chemistry students. Students effectively differentiated between strong and weak protein−protein interactions (median correlation coefficient between observed and expected results = 0.88) and demonstrated keen interest in the assay and concepts. Students were asked to then design and conduct a variation of the ELISA to test their own hypotheses regarding various experiment parameters to great success. Image acquisition for assay colorimetry was identified as a potential area of improvement. We have shown that this experiment is accessible to high school students both fiscally and academically and can be a fun and effective tool to apply their knowledge of intermolecular forces within the context of proteins. We have shown that the experiment could also be implemented in an undergraduate laboratory setting to allow for advanced inquiry into protein−protein interaction quantification and data analysis. This experience helps students at a variety of academic levels make conceptual connections across the fields of chemistry, physics, and biology. KEYWORDS: Proteins/Peptides, High School/Introductory Chemistry, Biochemistry, Bioanalytical Chemistry, Hands-On Learning/Manipulatives, First-Year Undergraduate/General, Laboratory Instruction, Public Understanding/Outreach

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autoimmune disorders,3 and antibodies detecting chorionic gonadotropin in a pregnancy test. The strength and selectivity of protein−protein interactions are dictated via the complex interplay of the chemical groups within numerous amino acid side chains, the polypeptide backbone, and post-translational modifications.4 Thus, the importance and diversity of protein− protein interactions make them a valuable component of curricula in chemistry, biology, and biochemistry. Our goal was to develop an experiment that would allow students at a variety of educational levels to apply their knowledge of the intermolecular forces to the potential effects on the strength

INTRODUCTION

The structure−function relationships of molecules are a key concept for students to understand when studying chemistry and biology at both the high school and undergraduate level. The majority of students are taught the fundamentals of intermolecular forces via small molecule interactions probed by changes in bulk properties such as changes in boiling point, surface tension, and vapor pressure.1 However, at both the high school and introductory college chemistry level, students are rarely exposed to how these chemical groups interact in the context of macromolecules. One important class of noncovalent macromolecule interactions is protein−protein interactions, which are ubiquitous in science and medicine. Examples include insulin/insulin receptor critical to metabolic signaling,2 tumor necrosis factor antagonists for treatment of inflammatory © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: May 5, 2016 Revised: February 1, 2017

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DOI: 10.1021/acs.jchemed.6b00323 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

mutations) on a protein−protein interaction or one that can be done without significantly expensive equipment. We have developed and implemented an ELISA-based protein−protein interaction experiment that is readily accessible to both high school and undergraduate classrooms to teach students how various chemical groups within a protein can affect the strength of its interaction with another protein. The experiment consists of an ELISA using a panel of protein ligand mutants with a ∼460-fold range of binding affinities for immunoglobulin G (IgG; i.e., an antibody). Students are able to perform the ELISA on the panel of protein mutants to quantitatively analyze the affinity of a protein with a known mutation in the hypothesized binding area. With costeffectiveness in mind, previously developed methods of camera phone colorimetry detection13,14 are used to bypass the use of a laboratory spectrophotometer. Using their results and knowledge of intermolecular forces, students are asked to speculate on the effect of certain mutations on the affinity of the protein− protein interaction. Students also completed a second inquiry for the ELISA experiment to address their own auxiliary questions by investigating the impact of one of many experimental parameters such as (1) temperature, (2) incubation time, (3) pH, and (4) concentration. The initial guided inquiry gives the students experience in common biomolecular experimental techniques and teaches them basic components of molecular biology and the effects that various chemical groups can have on intermolecular interactions in the context of large biomolecules. The second experiment, an open inquiry application of the experiment, allows students to apply the knowledge they learned from the initial experiment to test various parameters of the ELISA as well as additional questions regarding the protein−protein interaction. This experiment can also be used in undergraduate biochemistry classes as a lesson on amino acids and protein−protein interactions and affinities, as the camera phone data acquisition can be quantitative enough to yield affinity values for the protein−protein interactions. If a spectrophotometer is available, it can be used in place of a camera phone for data acquisition (differences between the two methods are discussed further). Additionally, several reagents within this experiment are common across many molecular biology experiments, which would minimize the costs specific to this experiment by sharing reagents across experiments.

of a complex protein−protein interaction. First, we focused our efforts in designing an educational unit for high school classrooms that complies with the Next Generation Science Standard (NGSS HS-PS1−3), which states that students will be able to “plan and conduct an investigation to gather evidence to compare the structure of substances at the bulk scale to infer the strength of electrical forces between particles”. We additionally wished to show that these experiments could easily be expanded in complexity to be used in undergraduate laboratories to rigorously investigate protein−protein interaction properties, as well as the effect of a wide variety of assay conditions, on biomolecular forces. Because introducing the concept of proteins and their interactions can be difficult for students at both a high school and undergraduate level, a hands-on exemplification of concepts in action could improve the students’ abilities to make connections between the properties of atoms alone and within macromolecules as well as learn concepts fundamental to molecular biology and chemistry. As beneficial as this experimental concept could be, implementation of protein−protein interaction experiments often requires expensive equipment and reagents as well as lengthy time spans. Numerous assays, such as enzyme-linked immunosorbent assay (ELISA),5 surface plasmon resonance,6,7 and flow cytometry,8,9 are used to evaluate the binding strength, or affinity, of the protein−protein interactions. Surface plasmon resonance and flow cytometry are highly effective, quantitative techniques but require expensive equipment. ELISAs present a potentially accessible technique and would provide students exposure to a technique that is heavily used in research and clinical laboratories. Briefly, in an ELISA, a target protein is immobilized in a test tube or sample well, either directly or indirectly via an additional molecule, and its binding partner is then added. Binding of this partner protein is monitored by another protein (typically an antibody), which specifically binds to the partner protein in a different location, and is fused to an enzyme. The amount of partner protein binding, which is a function of protein concentration and affinity, is proportional to the enzyme concentration. The enzyme concentration is then quantified by measuring the amount of product generation upon substrate addition via absorbance at a specific wavelength. This experimental technique has been previously modified for use in undergraduate laboratories by detecting small molecule interactions using multiwell plates10 and microfluidic chips11 and analyzing unknown concentrations of antibodies in test tubes.12 However, these protocols are generally focused on practicing experimental technique and data analysis rather than direct inquiry about the effects of specific amino acids on a known protein−protein interaction. Moreover, the majority of ELISAs are designed to detect the presence of a specific molecule, but the details of the protein−protein interaction between the main binding partners are generally not known in enough detail. This hinders the ability to discuss the effects of individual amino acid side chains (i.e., chemical groups) on the overall binding strength. Furthermore, different versions of the same binding partners are generally not available to interrogate the effect of different amino acids on binding strength. These protocols also require specialized materials (microchips, specialized and expensive antibodies) and equipment (centrifuges, plate readers, spectrophotometers) for implementation and data acquisition. To our knowledge, there is no current experiment available that tests the effects of variations in chemical groups within a binding site (i.e., single-site

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HAZARDS Care should be taken when using the hot plate to warm the substrate solution, and good laboratory practices should be followed (see Supporting Information for details). None of the reagents poses acute or chronic hazards when used properly. Refer to reagent-specific material safety data sheets for further information. All reagents can be handled with gloves and eye protection.

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RESULTS

Assay Development

The main hurdles in developing an accessible, educational ELISA for learning about protein−protein interactions were: (1) identifying an appropriate pair of binding proteins and associated reagents that are functional, interesting, educational, and accessible; (2) optimizing the logistics of the ELISA, which often includes lengthy incubations, in a brief classroom setting; (3) enabling efficient quantification of product formation with B




subdomain of the much larger human fibronectin protein that researchers have used to engineer several different versions that bind strongly to various partners15,16 including the IgG proteins originating from rabbits (rabbit IgG).17 We chose this interacting pair for several reasons: (1) the materials are relatively inexpensive as IgG can be purchased for $174/50 mg plus shipping (only 0.65 μg of which is needed per student or group), and Fn is efficiently produced recombinantly; (2) IgG is large enough that it can be nonspecifically adsorbed to the microwell plate while sufficient binding site accessibility is retained, which eliminates the time and cost of indirect immobilization via an additional molecule; (3) Fn is recombinantly produced with a His6 peptide tag, which enables efficient, inexpensive detection with an anti-His6 antibody conjugated to horseradish peroxidase (HRP) enzyme; (4) Fn mutations that modulate the strength of the protein−protein interaction should be readily identifiable; and (5) structural data are available for both IgG and Fn, albeit not for the precise mutants used in the assay, though this is the subject of ongoing efforts. The molecular components for the experiment (suppliers, catalog numbers, and costs in Supp. Table 1) are (1) rabbit IgG, (2) milk protein, (3) FnR0.6.2, (4) anti-His6 antibody−HRP conjugate, and (5) tetramethylbenzidine (TMB) substrate, which is oxidized, by HRP, into a blue product (Figure 2A). The typical format for an ELISA is done within a day, and we have also included a protocol for adapting this specific experiment to a 3−4 h window that is appropriate for undergraduate laboratories. Incubation times for specific steps can depend on the experimental conditions, and we have noted this in the protocol supplied in the Supporting Information. For use in a high school setting, we designed an experimental workflow to fit into a 45 min daily class within 1 week (Figure 2B). On day 1, rabbit IgG is added to several wells of a 96-well microplate and allowed to adsorb at 4 °C overnight. On day 2, unadsorbed IgG is removed, the wells are washed by phosphate-buffered saline, and milk protein is added to block any surface areas that were not coated by IgG to prevent nonspecific adsorption of Fn or anti-His6−HRP conjugate. On day 3, unadsorbed milk is removed, the wells are washed with PBS, and Fn (or buffer-only negative control) is added to the well and allowed to incubate overnight at room temperature. During incubation, the Fn and IgG are able to noncovalently interact to form a complex. The amount of complex formed varies based on Fn concentration and the affinity of the Fn IgG interaction. Notably, the experimental protocols on days 1−3 consume less than 20 min of class time, which allows for

available or low-cost equipment; and (4) validating the ability of inexperienced students to successfully perform the experiment. To find an appropriate protein binding pair, we looked at the numerous proteins that have been developed in our lab to bind strongly and specifically to various other protein targets. From this large pool of candidates, FnR0.6.2 and its target protein, rabbit IgG, were hypothesized to be the most viable for the assay (Figure 1). The Fn protein is a 94 amino acid

Figure 1. Fibronectin structure. (A) The solution structure (1TTG18) of wild-type tenth type III domain of the fibronectin protein (abbreviated Fn) is depicted in ribbon format. This Fn domain is a 94-amino acid β-sandwich protein with three solvent-exposed loops that can be heavily mutated to generate new binding function. The mutated sites used to originally engineer IgG binding are highlighted in blue. Site Y78, which is mutated to various amino acids to modulate binding affinity for the classroom experiment, is highlighted in red. (B) Size comparison of the Fn3 structure to the proposed binding partner, IgG (1IGT19). Because of its large and diverse yet stable folded structure, the IgG molecule allows for a diverse range of possible protein−protein interactions with other engineered ligands.

Figure 2. ELISA approach. (A) Components of the ELISA are presented. (B) The schedule, for a 45 min daily class, is shown. Component and protocol details are provided in the text and Supporting Information. C




discussion of concepts and auxiliary activities related to the experiment. Conversely, day 4 is a full experimental day. Unbound Fn is removed, wells are washed with PBS, and bound Fn is labeled by addition of anti-His6−HRP antibody− enzyme conjugate. Additionally, for adaptation to an undergraduate class, days 2−5 could be completed within a 3−4-h window (Supporting Information: Single Day Protein ELISA) by using either a camera phone or plate reader for quantification and requiring only the first step of rabbit IgG absorption to be done by the instructor the previous day. Both methods provide comparable data quality (Figure 5A). The amount of HRP present, which will be proportional to the amount of bound Fn, is quantified by addition of TMB substrate. Substrate conversion is measured by taking a photograph (e.g., using a mobile phone) of the microwell plate after addition of substrate. On day 5, the photograph is quantitatively analyzed using a color-picking application through a compatible web browser. If the experiment is conducted in a single day undergraduate lab, the use of the plate reader would eliminate this time required for image analysis. The experiment, as performed in both a single day and 4-day format by the teacher and a graduate student in preparation for classroom use, effectively differentiated between the highaffinity FnR0.6.2 and the buffer-only negative control (Figure 2B).

Figure 3. Fibronectin mutants of varying affinities. (A) Titrations of Fn mutants in the ELISA assay demonstrate differential affinity. Fn mutants, in which the amino acid at site 78 is indicated via color in panel B, were titrated via the single-day ELISA protocol (see Supporting Information for full protocol) by graduate student and instructor. Data shown are representative of a single titration of each Fn mutant (◊) and theoretical binding fraction. Each isotherm was calculated based on the Kd determined from three or more titrations (see Supp. Figure 1 for extended data). (B) The amino acid side chain at site 78 is shown along with the equilibrium dissociation constant calculated from the titration.

Identify Mutants with a Range of Binding Affinities

To widen the breadth of the experiment and facilitate the discussion of protein−protein interaction forces, we sought to include FnR0.6.2 mutants with a range of affinities for IgG. We desired the mutations to all occur at the same Fn site for simplicity in discussing the impact of the various chemical groups within the amino acid side-chains on the protein− protein interaction. We chose to mutate a tyrosine residue at site 78 (Y78) in the third loop of Fn (Figure 1) for several reasons: (1) the site is near the middle of the evolved binding region, which renders it likely to be impactful for binding; (2) deep sequencing of a diverse population of evolved binding ligands suggested that Y78 was impactful for binding;17 (3) tyrosine residues are often impactful for protein−protein interactions;20 and (4) tyrosine can act via several phenomena important for protein−protein interactions (hydrogen bonding, cation−π interactions, hydrophobic effect, and van der Waals interactions; and its rigidity enables discussion of conformational entropy),4 which permit discussion of these effects to whatever level is appropriate for the particular classroom. For example, students at the high school level can discuss differences in amino acids in a simplified manner by grouping amino acids into “hydrophobic” or “hydrophilic” categories, but more advanced undergraduate students can be asked to expand on the gradient nature of hydrophobicity by each amino acid and their effect on binding and also discuss intramolecular interactions of the mutated amino acid and its neighboring residues within the binding portion of the Fn protein. Experimental methods for gene synthesis and protein production can be found in the Supporting Information. Once produced, the affinity of each mutant was determined by titrating Fn concentrations in the ELISA. Analysis of the parental tyrosine and four random mutations yielded a good range of affinities (from 1.5−696 nM) and diverse chemical structures for the students to interrogate (Figure 3). Thus, an ELISA performed at a single concentration of Fn, for classroom

simplicity, can yield a range of binding signal to highlight the differences in protein−protein interaction strength. For example, at 100 nM Fn, the signals yielded at equilibrium will reach 99% of the maximum for parental tyrosine, intermediate signal for isoleucine and lysine (64% and 54%. respectively), and low but detectable above background for serine and glycine (19% and 13%, respectively). If desired, multiple concentrations can be used to accentuate particular differences. Classroom Implementation

The experiment and supporting lesson plans were implemented in a three-week biochemistry unit to demonstrate the impact of molecular structure and intermolecular forces in protein− protein interactions. The unit was implemented at the end of the semester in two honors chemistry classes (32 students per class, predominantly juniors and seniors) at Shakopee High School. In the first week, students were introduced to proteins, amino acids, and various examples involving intra- and intermolecular forces with biomolecules. Students were also tasked with a 3D-Toober activity to illustrate how amino acids dictated protein folding21 and a prelab activity to hypothesize about the structure−function relationships of the different amino acids and to predict their effects on binding affinity. In week 2, students performed the experiment, in groups of two to four students, to compare binding of the parental FnR0.6.2 (Y78) and four mutants (I, S, K, and G) at 100 nM as well a bufferonly (i.e., 0 nM) negative control (protocol in Supporting Information). During the third week, students were asked to plan and conduct an investigation to test their hypothesis on the effects of various ELISA experimental conditions. D




Figure 4. Student group results. Twenty groups of students performed the 5-day ELISA protocol (see Supporting Information for full student instructions) using 5 Fn mutants at a concentration of 100 nM each. The students’ results (y-axis of 4(A) and 4(B)) were compared to the predicted results (x-axis of 4(A) and 4(B)). (A) Averaged raw data from students selecting the green channel value in three locations of the well shown on the Y-axis versus the theoretical fraction of maximum allowed fibronectin binding (f), where [Fn] is the molar concentration of the Fn (100 nM), and Kd is the affinity for each mutant (shown in Figure 3). Because of the blue color of the reporter molecule, as f bound increases, the blue color deepens, which decreases the green channel value. Each color denotes a separate student group. (B) Corresponding absorbance calculations from data in panel A. Notably, this calculation introduces additional error into each data point by normalizing to the blank (no Fn) well. (C) The correlation coefficients for the results in panel A are depicted for each group and as a box-and-whisker plot denoting the 1st, 2nd, and 3rd quartiles as well as the standard deviation. The majority of student groups successfully identified the correct trend of increasing color with increasing f bound.

Results were very good as most groups observed the expected correlation between ELISA signal and binding affinity (Figure 4). The median correlation coefficient was 0.88. Notably, the strength of this correlation was driven by consistently high signal from the parental Y78 Fn (90% of groups had maximal signal from Y78) and consistently low signal from the buffer-only control (85% of groups had minimal signal from this control). Because of the similar affinities, the students ability to differentiate between mid affinity (I, K) and low affinity (G, S) was modest. The median correlation coefficient between experimental and expected signals for these four samples was 0.71 (mean = 0.48). While the results were largely favorable, we also sought to improve the ability to differentiate similar affinities. Two hypotheses for these results are inconsistent image quantification (discussed below) and suboptimal Fn concentration. A reduced Fn concentration theoretically improves the ratio of signals between mid- and low-affinity mutants but also reduces the low-affinity signal to approach background. Notably, experimental noise increases at low signal. After the first experiment, students were given the opportunity to perform a follow-up experiment to test an additional hypothesis about intermolecular forces in proteins. These hypotheses included what would happen if changes in pH, concentration, buffer concentration, and temperature were made. In general, the students showed results that could be plausibly explained, with only one group having results that were not explained outside of their reported execution mistakes. The results were also very consistent across the different groups doing identical experiments, including the groups that varied pH, temperature, rabbit IgG concentration, and Y78 concentrations. Most students supplied logical hypotheses and conclusions for their experiments; however, more time to study the initial experiment would have eased interpretation of their new results. These results show that the experiment is amenable to varying conditions for students to explore additional hypotheses. The results and their interpretation were aided by an interactive forum between the high school students and

graduate students in the Hackel lab (Supporting Information) as well as in-person graduate student experimental oversight. Students were successful in proposing plausible explanations for the changes (or lack thereof) with their various conditions based on their knowledge of proteins and intermolecular forces. For the few experiments that were conducted by more than one group, results were qualitatively consistent. Results of a postunit survey show that students felt they possessed a broad understanding of the material but lacked confidence in some experimental details. Also, they did not feel prepared to design a follow-up experiment without adequate time to digest the initial results. This indicates that added time for discussion of results and additional activities could improve the students’ understanding of the experiment. The exploratory experiment should be done only after the students have demonstrated their understanding of each experimental step and protein−protein interactions. Cost Summary

Supporting Information provides a cost analysis for each reagent in bulk as well as the cost normalized to the amount used per 10-group class. When purchased in bulk, reagents and supplies specific to the experiment cost $606.82 including domestic shipping. However, if distributed among numerous classes within a school or area, when the small amounts of the reagents needed per group are factored in, the cost is substantially lower ($16.89 for experiment specific supplies per 10-group class) and will cover a minimum of 12 classes, with several reagents lasting up to hundreds of classes. When stored properly (select reagents require frozen storage, see catalog information in Supp. Table 1), these reagents last beyond one year. This allows for teachers to use the reagents for several semesters of experiments, with the number of classes each reagent can be used for also noted in Supp. Table 1. Additionally, several of the dry supplies may be purchased and shared between classes and experiments, and given the proper planning, could cost less per class. Though the most expensive reagent is the rabbit IgG, it can be used for over 1500 classes and can be stored for several years’ time. The main limiting E




multiple dilutions of a static dye (methylene blue); all five students then each analyzed all five images. Variance among the photos (relative standard deviations: 18 ± 3% for signal/ concentration slope and 9 ± 4% for linearity) was greater than variance among analyses for a single photo (8 ± 4% and 5 ± 3% for slope and linearity, respectively); that is, image analysis was more consistent than image capture. Moreover, analysis of the linearity of the signal versus concentration plots indicates larger error at low concentrations (Figure 5B), which likely resulted from image artifacts such as shadows or well edges. Thus, improved image acquisition, especially at low product formation rates, will be emphasized in future implementations.

experiment-specific supply on a per-experiment basis is that of the 96-well plates. The main capital expense for required materials is that of the micropipettes that are required for the accurate measuring of buffer components and Fn dilutions for incubation, but are also a capital investment that can be used across various biology and chemistry laboratories. Image Acquisition and Analysis

Acquiring product formation data via a simple camera (mobile phone or stand-alone digital camera) rather than a laboratorygrade spectrophotometer greatly aids accessibility of the experiment. However, it is critical that the camera approach provides consistent results to provide proper experimental feedback to the students. Camera colorimetry was recently shown to provide consistent calibration curves for multiple solutions in cuvettes and test tubes.13,14 Herein, we assessed the consistency of results in multiwell plates and the impacts of photo collection and analysis. To assess the effectiveness of the phone colorimetry to calculate absorbance, the absorbance at 455 nm measured by the plate reader after addition of stop solution was compared to the absorbance calculated by phone colorimetry prior to addition of stop solution (Figure 5A). The data show similar results when consistent image acquisition is implemented.

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DISCUSSION The experiment was developed to encourage wide adoption at the high school level along with consideration for undergraduate laboratories. The initial most costly reagents, rabbit IgG and Anti-His6-HRP ($313), provide sufficient material for over 384 experiments, and the most limiting reagents could be conservatively used for ∼12 classes (individual reagent breakdown supplied in Supp. Table 1). Thus, implementation across several classes or schools makes the per-class investment reasonable. Moreover, most of the reagents can be stably stored for future semesters. In addition, in acknowledging the large excess of most materials, one could envision a commercial kit with appropriately sized reagents being made available; note that the per-experiment cost is $16.89, with the main per class expense being the well plates. Fibronectin domains were the only noncommercial reagent but were selected for several reasons including ease of production. The plasmids are readily available from the Hackel lab or Addgene (www.addgene.org). A brief collaboration with a local academic or commercial biotechnology laboratory would be able to provide sufficient material for numerous experiments with minimal effort. For undergraduate laboratories and collaborating scientists, the production and purification protocol has been provided in the Supporting Information. All mutants generate multiple classes worth of fibronectin domains per 1-L batch. Such a collaboration should be mutually beneficial (such as the collaboration that led to this experimental development). This provides an opportunity for university faculty and graduate students to participate in meaningful STEM outreach to bring unique research experiences into high school classrooms. Funding for such programs is available through various government and privately funded organizations. In addition, a limited number of protein aliquots are available from the Hackel lab at the University of Minnesota. The experiment was highly successful in engaging students and aiding learning. Concerning experimental execution, students did well despite having no prior experience using either pipettes or the colorimetry assay. Students were also tested on their knowledge of various intermolecular forces common to proteins. On a final assessment, students were asked to identify three types of intermolecular forces; 79% of students correctly identified ionic interactions, 85% of students correctly identified hydrogen bonding, and 66% correctly identified hydrophobic interactions (see Supporting Information for questions and answer breakdown). Students were less successful identifying which amino acids were hydrophobic or hydrophilic, with 47% correctly identifying the hydrophobic and 32% correctly identifying the hydrophilic amino acid from a given set. Notably, students did very well on questions where the schematic depicted an intermolecular interaction and did

Figure 5. Image acquisition. (A) Comparison of absorbance measurement from plate reader and from camera phone colorimetry analysis. Absorbance via phone colorimetry was calculated from the green channel intensity A = −log(I/I0), where I is the average green channel intensity, and I0 is the average green channel intensity of the blank. (B) Methylene blue was added to a 96-well plate at 0, 2.5, 10, 25, 50, 100, and 200 μM. Images were acquired via camera phone. The deviation between the measured signal and the signal expected based on the linear fit of each model student’s data is indicated. A representative image of the dye dilution is included for visual reference.

While the majority of groups observed a strong correlation between affinity and ELISA signal, we sought to improve the ability to differentiate similar affinities and to explore the cause of poor correlations for some groups. To assess if inconsistencies resulted from discrepancies in image acquisition or analysis, we evaluated the consistency of both steps. Five “model” students were asked to take photos of wells with F



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CONCLUSION An experiment was developed that could help students learn about the structure−function relationship of chemical groups and intermolecular forces in the context of protein biochemistry at the high school or undergraduate level. An assay that could be completed within the time constraints of a daily high school class was performed successfully by the students. Further analysis of error in the data acquisition was investigated, and it was determined that when given a specific set of instructions, students were able to reproduce similar trends with colorimetry analysis as a direct absorbance measurement. A survey of the students found that their overall experience was positive; however additional time for “set up” activities and analysis following the experiment is highly suggested. Further suggestions on activities were made for implementation in a high school classroom as well as modifications to the experiment and overall unit to produce students with a strong fundamental basis in the chemistry involving biomolecules. As it stands, it is a robust and useful educational tool to teach students about intermolecular forces within complex biomolecules, as well as experimental design and execution, at a variety of education levels.

worse on identifying properties of an individual molecule (in this case, an amino acid). Overall, students were able to successfully identify the types of intermolecular forces within proteins; however, the gradient nature of hydrophobicity of a molecule made it more difficult for students to correctly differentiate between hydrophobic and hydrophilic amino acids. When asked whether they would suggest doing the experiment in following semesters, 95% of students supported performing the experiment again, with their main suggestion being to spend additional time before and after the experiment discussing the new material. Indeed, the experiment will be implemented in future semesters along with considerations of several modifications, offered below. Moreover, two additional instructors will be implementing the experiment, and we hope to facilitate adoption of the experiment by other interested instructors within the area. The amino acid mutants used provide a broad range of affinities, to assess the impact on the binding assay, and a broad range of chemical diversity: aromatic (Y), hydroxyl (S, Y), aliphatic (I), and basic (K) and both large, medium, and small. Additional mutants would permit direct comparisons of certain functional groups, which could be incorporated in further developments. For example, comparing the parental Y78 to F78 would directly assess the impact of tyrosine’s hydroxyl group. In addition to the FnR.0.6.2 gene, all 19 possible mutants at position Y78 are being made available in a ready-to-produce format on Addgene and can be purchased as a set for a reduced cost. Image acquisition was identified as the primary source of inconsistency in the results. The protocol has been modified to emphasize the importance of consistent and accurate photography. Further implementations will also allow students time to practice image acquisition on a static blue dye at various dilutions within the ELISA plate and supply a “picture station” that can be set up to provide an optimal lighting scenario for the specific classroom. For undergraduate laboratories or classrooms with fewer budgetary or time restrictions, alternative methods of quantification, such as a plate reader or LED imaging setup,22 could be used. Additionally, replicates could be readily performed in adjacent wells to measure reproducibility (and facilitate education on this concept). The instructor can decide if these benefits are worth the time and reagent costs. Notably, the experiment was effective with single wells for each mutant, which is time- and cost-efficient. The timing worked well with regard to performing experimental steps in a comfortable manner. Notably, the experiment could be adjusted to accommodate block scheduling or single-day completion for college courses. Student feedback indicated that the class activities−protein model, prelab worksheets, and intermolecular lessons in the week prior to the experiment were highly valuable. Deeper discussion of the experimental steps and practice ELISA activities were suggested to improve understanding. For adaption to undergraduate curricula, expansion of the experiment to multiple concentrations, mutants, and incubation conditions could be useful for teaching concepts of binding kinetics and equilibria as well as basic biochemistry assays. One could also foresee students having to design an experiment to identify a specific mutation based on known Kd values and discuss fundamental energetic rationale behind the effects of various mutations on the binding energy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00323. Questions and responses from interactive forum; experimental protocols; materials and methods for ELISA and production and purification of FnR0.6.2 mutants; suggested reagents and costs; suggested activities and student handouts with instructor notes; titration data; student exam questions and score breakdown for assessment of pedagogy; preunit assessment quiz (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Benjamin J. Hackel: 0000-0003-3561-9463 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported partially by the National Science Foundation through the University of Minnesota MRSEC under the RET program (Award No. DMR-1263062). The authors thank R. Lee Penn and Cassandra M. Knutson for helpful discussions.

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REFERENCES

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