The recently published ASBMB report on biochemistry and molecular biology education systemically catalogued the foundational concepts, theories, skills, and outside knowledge in which all undergraduate biochemistry and molecular biology majors should be proficient [1-3]. Amongst the ideas championed by these reports were the focus of biochemistry and molecular biology education on big qestions in science, the integration of classroom material with larger societal issues, and the instruction of students in scientific skills [2, 3]. First semester biochemistry courses traditionally cover a wide range of topics from basic building block structures to complex biochemical phenotypes in loosely connected vignettes. Biochemistry textbooks reinforce this arrangement with discrete chapters for each concept and distinct biochemical examples for each topic interspersed. This disjointed arrangement of topics makes it difficult to construct a cohesive story about how biochemistry addresses big picture questions in science and how biochemistry impacts the larger society.
To provide a big picture framework for student integration of biochemistry topics, to get students excited about the role of science in society, and to foster scientific skills, I have developed a series of classroom exercises that connect diverse topics from the first semester biochemistry curriculum together around a central theme of HIV protease (HIV PR) and its inhibitors (Fig. 1). HIV PR is arguably the most studied enzyme of the twentieth century and has served as a model for enzyme structure, mechanism, structure-based drug design, and protein evolution [4-9]. HIV PR is a small, dimeric aspartic protease that plays an essential role in the viral life cycle, cleaving the viral Gag and GagPol precursor proteins into the mature viral proteins [4, 5, 9]. The necessity of these cleavage reactions for viral maturation has made HIV PR a key therapeutic target with 10 currently FDA-approved HIV PR inhibitors [4, 5].
HIV PR also serves as a perfect model for big picture connections in the biochemistry classroom with its well-studied biochemistry, its clinical applications, and its global health connections. In addition to connecting diverse biochemistry topics from thermodynamics, amino acids, protein structure, protein purification, ligand binding, enzymatic catalysis, and enzyme inhibition, these classroom exercises also involve students in the primary biochemical literature, in analyzing research data, in critically evaluating biochemistry research, and in accessing biochemical resources (Table I). Herein, I describe the basic framework for each of these exercises, their classroom implementation, and their connection to the traditional biochemistry lecture material.
Overview of Exercises
Within the first semester biochemistry course, I introduce the topic of HIV/AIDS and its importance to global health on the first day of class. This sets the framework for the course, provides an introduction to the viral life cycle of HIV, and defines the role for HIV PR within the life-cycle of HIV. The course then continues along a fairly standard biochemistry lecture trajectory beginning with introductory topics like acids/bases, thermodynamics, and intermolecular forces, and continuing into proteins from simple amino acid structure to complex enzyme kinetics and mechanisms. Each of nine key topics within the biochemistry lecture is then connected to the big picture of HIV PR and its inhibitors using classroom exercises and discussions (Table 1). For each of these exercises, an entire lecture period of 50 minutes is committed to the exercise, requiring 9 days of lecture to complete all of the assignments. A description of how the 50-minute classroom time is separated into individual work, group work, and classroom discussion is provided within the detailed descriptions below. For each exercise, students are provided in class with detailed handouts and learning goals for all nine exercises (Supporting Information Document 1). Within the 40-student lecture course, students are allowed to choose their own groups of 3–5 students for discussions and exercises.
Table 1. Integration of the HIV protease exercises into a first semester biochemistry course
The nine HIV PR exercises were designed to highlight foundational biochemistry topics and to incorporate diverse active-learning styles into the classroom (Table 1). Exercises are conducted immediately following the introduction of the required background material in standard lecture format. The organization of the material in relation to common biochemical textbooks and the prerequisite material for each exercise is outlined in Supporting Information Document 2. Student learning on the exercises is assessed using exam questions that parallel the HIV PR assignments, but apply the material to new circumstances. Detailed assessment questions covering all of the diverse topics are provided in Supporting Information Document 3.
The following subheadings summarize the basics of each exercise, the goals of the exercise, the classroom instruction, and the time required for each exercise.
Outline of Classroom Exercises
Exercise 1: Energetics of HIV PR Inhibition
Thermodynamics, intermolecular forces, and the hydrophobic effect are central topics in biochemistry that are introduced as discrete topics in the beginning of the semester. The first HIV PR exercise requires that students read only the introduction and conclusion sections to a research article entitled, “HIV-1 protease inhibitors: enthalpic versus entropic optimization of the binding affinity” . Students then work in groups to determine the factors that control the energetics of HIV PR binding to its inhibitors, including how the energetic favorability of the HIV PR interaction with the first generation PR inhibitors is determined solely by a favorable entropic contribution (hydrophobic effect) . Students then wrestle with the hypothesis that shifting the balance of the entropic and enthalpic contributions to inhibitor binding could combat the growing resistance of HIV PR to first generation PR inhibitors. This hypothesis is eventually confirmed with the discovery and testing of the most recent inhibitor darunavir and the articles for exercises 7–9 bring this discussion full circle [12, 13]. As most students have not dealt with protein ligand binding previously, a general introduction to the topic is first provided, including simple pictorial representations of protein and ligand binding. This article was chosen for the first exercise because it creates an overall story arc to the semester, but similar foundational topics could be reinforced with a discussion of the differential solubilities of HIV PR inhibitors in octanol/water . This initial exercise requires 5 minutes of introductory lecture, 35 minutes of student small group discussion, and 10 minutes to summarize the discussion.
Exercise 2: HIV PR Inhibitor Structures
Initial HIV PR inhibitors were peptidomimetics that were directly modeled on the peptide sequences of HIV PR substrates [4, 5]. The first generation inhibitors contain a full peptide backbone with the insertion of a hydroxyethylene bond in the middle that mimics the transition state and inactivates the catalytic aspartates of HIV PR (Fig. 2) . Second generation inhibitors no longer contain a full peptide backbone, but maintain similar chemical moieties at each position in the inhibitor (Fig. 2) . For this exercise, the structures of the 10 different FDA-approved HIV PR inhibitors are presented to the students. Students then analyze the structures of the inhibitors, identify the structures of different amino acids, look for the standard peptide bond architecture, and find the reoccurring hydroxyethylene bond. The larger classroom discussion centers on the role of the hydroxyethylene bond, the characteristics of a good drug, and the general patterns identified in HIV PR inhibitors. This exercise requires 35 minutes of student small group analysis/discussion and 15 minutes of summarizing classroom discussion.
Exercise 3: HIV PR Cleavage Sequences
Building on the analysis of the inhibitor structures and the presence of specific amino acids (exercise 2), exercise 3 presents students with an alignment of 10 polyprotein cleavage sequences recognized by HIV PR . Students work in groups to analyze the preference of HIV PR for specific amino acids at each position of the cleavage sequence. Based on this analysis, students compare the levels of conservation at each position and develop a general motif for HIV PR cleavage sequences. This exercise emphasizes the general grouping of amino acids based on their structures, their relative properties, sequence alignment, and sequence motifs. This exercise also requires 35 minutes of student small group analysis/discussion and 15 minutes of summarizing.
Exercise 4: HIV PR Structure and the Protein Data Bank
With over 500 structures of HIV PR available in the Protein Data Bank (PDB), HIV PR serves as a good model for student exploration of the PDB and introduction to three-dimensional protein visualization software [5, 9]. This exercise involves students in the PDB by searching for structural information in a PDB file and by manipulating the three-dimensional visualization of an HIV PR structure. Specifically, the exercise looks at a co-crystal structure between HIV PR and amprenavir, a second-generation HIV PR inhibitor . The exercise reinforces general topics about protein structure, including levels of protein structure, protein domains, and protein structure determination methods. Given the necessity for a computer to access the PDB, this exercise has been conducted as an in-class and out-of-class exercise, and requires about 25–30 minutes to complete.
Exercise 5: HIV PR Inhibition and Dynamics
To continue their involvement in protein structure and to access new resources for protein structural comparison, exercise 5 uses Proteopedia to provide another viewpoint of HIV PR structure and inhibition [18, 19]. The advantage of Proteopedia is that it systematically takes students through different HIV PR structures and highlights the key features of each structure [18, 19]. The Proteopedia page for HIV PR focuses on the location of inhibitor binding and shows the structural adjustments to HIV PR upon inhibitor binding. HIV PR has two structural flaps that must clamp down on its cleavage sequences and inhibitors to facilitate tight binding and inhibition . The movement and dynamics of flap movement are modeled on the Proteopedia page. The Proteopedia page and accompanying exercise reinforce foundational topics and skills on protein structure and function, three-dimensional visualization of protein structure, and protein stabilization and dynamics. Similar to exercise 4, this exercise can also be conducted as either an in-class or out-of-class exercise, requiring 20–25 minutes to complete.
Exercise 6: HIV PR Purification and Analysis
Students begin exercise 6 by considering the basic chemical and physical properties of HIV PR that could be used to separate it from other cellular proteins [7, 9]. Students then dissect the experimental procedures and results sections for a research article describing a four-step purification of HIV PR (“Purification of Recombinant HIV Protease”) . This purification of HIV PR utilizes four different chromatography techniques (anion exchange, hydrophobic interaction, cation exchange, and size exclusion), clearly labels the locations for HIV PR elution from each column, and analyzes the final purification using SDS-PAGE and simple activity measurements . The classroom discussion centers on the relationship between the purification methodology and the physical characteristics of HIV PR, the logic of each purification step, and the conformation of the correct purification of HIV PR using SDS-PAGE. The entire exercise requires 40 minutes of student group work and 10 minutes of summarizing discussion.
Exercise 7: HIV PR Inhibitor Binding
To prepare for exercise 7, two articles are assigned that relate the binding affinity of different PR inhibitors to their ability to resist inactivating HIV PR mutations [12, 13]. Together these articles detail how darunavir (the newest HIV PR inhibitor) with its significantly improved enthalpy of binding and increased binding affinity is the answer to the hypothesis that was posed in exercise 1 (Fig. 2) [12, 13]. For class, students are broken off into eight groups of 4–5 students and assigned a figure or table from one of the two articles. Each group is then given a list of questions to answer about the figure or table and to present to the class. The two articles and accompanying classroom discussion reinforce foundational topics including protein structure, ligand binding, equilibrium versus kinetic constants, and thermodynamics. The groups are given 10 minutes to prepare for their presentation and 3–4 minutes per group to present each figure for a total of 40 minutes of class time for presentations.
Exercise 8: HIV PR Enzyme Mechanism
Instead of directly lecturing about the mechanism of peptide bond hydrolysis by HIV PR, a review article is assigned that provides a detailed description of the HIV PR mechanism . Throughout the article, multiple proposed HIV PR hydrolysis mechanisms are discussed and evaluated . Students are then asked to examine the different mechanisms and to look for similarities and differences in the mechanisms. The article then provides a consensus mechanism and applies this mechanism to explaining how the hydroxyethylene bond in HIV PR inhibitors mimics the transition state for peptide bond hydrolysis . The classroom time is split between 30 minutes of student work drawing and evaluating the mechanisms and 20 minutes discussing the similarities/differences in the mechanisms. This exercise draws on lecture material related to basic enzyme catalysis, acid/base properties of amino acids, transition state theory, and experimental design.
Exercise 9: HIV PR Kinetics and Inhibition
The final exercise serves as the culmination to the lecture material on proteins and enzymes and pulls together many of the major topics from exercises 1–8. For this exercise, students are given data from multiple research papers that measured HIV PR kinetics and inhibition and are challenged to use this material to solve biochemical problems [21, 22]. The problems are designed to reinforce topics covered in lecture on enzyme kinetics, inhibition, ligand binding, and thermodynamics while maintaining the same general darunavir framework as exercises 1, 2, 7, and 8. The students work in groups on the problems for 50 minutes. Problems are checked throughout as the instructor rotates around the classroom asking students questions about their work.
In addition to providing a coherent and big picture framework for first semester biochemistry material, the nine exercises also dedicate classroom time to strengthening student's general scientific skills . The exercises focus on the reading, interpretation, and discussion of primary and secondary literature exercises, analysis of scientific data, accessing scientific resources, and presenting scientific results. The involvement in the primary literature begins with exercise 1, where students read only the introduction and conclusion sections and culminates in exercises 7–9, where students analyze figures and tables and provide a big picture comprehensive explanation of HIV PR inhibition. The exercises also stress the importance of data analysis, proper interpretation of figures and tables, and technical discussion of research articles in small group and large classroom settings. Finally, students gain experience with computational biochemical resources including the PDB, Expasy, JMol, and Proteopedia.
Refinement of the Exercises Based on Student Feedback
This classroom strategy of integrating foundational topics around the central theme of HIV protease has been implemented for three separate years. In the first iterations, the lack of diversity in student learning assignments and the lack of previous student experience in reading and analyzing primary literature were identified as issues detracting from student learning and engagement. To address these issues, the current assignments introduce more diverse active learning styles, including focusing on small group work, student presentations, collaborative problem solving, and online biochemical resources. The current course design has also been adapted to provide a ramped introduction to the primary literature and to gradually increase students' level of involvement in the primary literature. Overall, the course design has been well-received by students with many students stating on course evaluations that they appreciated seeing the real-life application of the course material. They also found that the HIV protease exercises made the classroom material more relatable and gave them a better understanding of the drug discovery process. Students still find reading the primary literature papers difficult, but stated that in-class discussions and assignments prepared them for the classroom evaluations and for critical analysis of scientific literature. Overall, the assignments were challenging to the students, but students commented that this helped facilitate a great active learning environment.
HIV PR has been a central model to enzymology, structure based drug design, protein dynamics, and three-dimensional structure determination [4-6, 9]. In this article, I have shown how HIV PR can also serve as a model for integrating the instruction of foundational topics in a first semester biochemistry course into a cohesive framework centered on a big picture scientific and societal problem (Fig. 1) . Through these exercises, students build on their classroom knowledge about protein structure and function to dissect the complex structure, enzymology, inhibition, and biological functions of HIV PR (Table I). Each of these exercises serves to reinforce central themes from lecture, to move students into active learning of the material, and to challenge students to integrate multiple topics into a coherent explanation. The exercises also incorporate training in important scientific skills, including reading primary literature, data analysis, collaborative problem solving, and scientific presentations. As these exercises cover foundational topics common to all first semester biochemistry courses (Fig. 1), these exercises should appeal to a broad audience of undergraduate students and should be highly adaptable to a variety of teaching styles and classroom sizes. From these exercises, students develop a broader sense of biochemistry and an understanding of scientific discovery, while learning about current treatments for HIV/AIDS.