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The creation of these assignments was supported by a National Science Foundation Course, Curriculum and Laboratory Improvement-Adaptation and Implementation Grant 0088129. The figures in this manuscript were produced in PyMol (http://pymol.sourceforge.net/).
The teaching of structural concepts plays a prominent role in many chemistry and biology courses. When it comes to macromolecular systems, a thorough understanding of noncovalent interactions lays a strong foundation for students to understand such things as protein folding, the formation of protein-ligand complexes, and the melting of the DNA double helix. The incorporation of computer-based molecular graphics into the biochemistry curriculum has given students unique opportunities in visualizing the structure of biological molecules and recognizing the subtle aspects of noncovalent interactions. This report describes a series of visualization-based assignments developed to facilitate protein exploration in a large biochemistry class. A large enrollment can present special challenges for students to benefit from hands-on use of visualization software. Three of the assignments are described in detail along with a description of an on-line teaching tool used to manage the assignments and to coordinate the student groups participating in these exercises.
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The visualization-based assignments described in this report were developed by the biochemistry instructor (J. R. C.) and a senior-level undergraduate student (D. W. H.). The paper is written from the perspective of the instructor and describes how molecular visualization has been incorporated into a large biochemistry class.
There has never been a more important time for students to learn about the structure and function of biological molecules. The use of computer-based molecular visualization programs has fundamentally changed how structural concepts are taught throughout chemistry and biology. The largest impact has been on the visualization of macromolecular systems, where programs such as RasMol (H. J. Bernstein, www.openrasmol.org) [1, 2], RasTop (P. Valadon, www.geneinfinity.org/rastop/), Protein Explorer (E. Martz, www.proteinexplorer.org) , Swiss-PDB Viewer (Deep View; N. Guex, A. Diemand, M. C. Peitsch, and T. Schwede, www.expasy.ch/spdbv) , and Kinemage (D. C. Richardson and J. S. Richardson, kinemage.biochem.duke.edu)  have become invaluable to many biochemistry and biology instructors [5–10].
The graphics programs can be used in the classroom by instructors or put in the hands of students to explore the three-dimensional structure of biological molecules . There are also many web-based tutorials that are excellent resources for instructors and students at all levels. The World Index of Molecular Visualization Resources (molvisindex.org) catalogs a variety of web-based tutorials and is an excellent place to start for anyone interested in incorporating visualization into their courses. Many of these tutorials are based on the Chime plug-in (MDL Information Systems, www.mdlchime.com) and allow a great deal of interactivity as the plug-in itself functions as a graphics program. One notable instructional website was recently described in Biochemistry and Molecular Biology Education and uses inquiry-based exercises combined with Chime-based tutorials to teach structure/function relationships in proteins . Many textbooks also have companion websites that offer students and instructors structural tutorials based on Chime. One example is Biochemistry in 3D (www.worthpublishers.com/lehninger3d/index.html), which accompanies the Lehninger Principles of Biochemistry textbook . Those interested in kinemages can find a great deal of information and supplements at the Richardson's Kinemage homepage (kinemage.biochem.duke.edu) and an undergraduate kinemage site maintained by Robert Bateman (orca.st.usm.edu/∼rbateman/kinemage).
I have previously described my use of molecular visualization to teach the structural nature of macromolecules [6, 11, 14]. One approach has been to focus on the different types of noncovalent interactions that stabilize proteins and protein-ligand complexes. Students dissect the protein systems to identify specific interactions between amino acid side chains, which include the underappreciated π-type interactions. In the two-semester biochemistry sequence at Murray State University, enrollment is much lower and students are asked to give oral presentations based on their search for noncovalent interactions. The large number of students in the one-semester biochemistry course (CHE 330, 50–60 students) makes it quite difficult to coordinate oral presentations by students or student groups. My previous experience has demonstrated to me that students benefit a great deal from being able to use visualization software to explore the structure of proteins. Therefore, I implemented the Protein Exploration Project in CHE 330, which consists of a series of assignments where students were asked to investigate the structure of proteins and protein complexes. Instead of giving oral presentations, students answered specific questions about the structural nature of proteins and protein-ligand complexes and the role of noncovalent interactions in these systems.
The participation of students in the assignments associated with the Protein Exploration Project was coordinated through Blackboard 5.0 (Blackboard, Washington, D.C., www.blackboard.com). Blackboard is a server software product used to deliver completely on-line courses or to enhance traditional courses, such as CHE 330.
This software allows an efficient exchange of information and electronic documents between students and the instructor. Blackboard enables an instructor to create student groups and establish a direct line of communication, which is extremely important to the success of group-based assignments. This software also provides the students with an on-line mechanism in which to chat and exchange documentation in a formal academic environment. This type of communication is important in facilitating the participation of all students in the group and mediating the dynamics of a diverse group of students. The instructor can monitor the Blackboard activities of the students and provide helpful suggestions to the groups or suggest that less active students become more engaged in the assignments.
Two of the most useful features in Blackboard were the External Links area and the Digital Drop Box. Many links to a variety of web pages were placed in the External Links area. For example, students could enter this area of Blackboard and have quick access to the Chime web page or visit the Chime-based tutorials used in the lecture portion of the course. The Digital Drop Box allowed the student groups to electronically submit assignments associated with the project and avoided the use of E-mail attachments and disk submission.
PROTEIN EXPLORATION PROJECT
To begin the project, student groups (3–4 members) were formed and trained to use Protein Explorer (PE).11 The training sessions were held in the Murray State University Chemistry technology-enhanced classroom. I used a 72-inch rear-projection SMART Board and the students used laptop computers in a wireless environment. The training sessions were vital to the success of this project as the students became comfortable with the software and learned to use it effectively to answer the questions in the assignments. Students also learned to recognize the structure of amino acid side chains in proteins when viewed in PE. Recognizing side chains in a computer-generated rendering of a protein can be challenging for students. The line, stick, or ball-and-stick representations of amino acids encountered in three-dimensional structures hardly resemble the 2D structures encountered in textbooks. The students also learned the fundamentals of PE such as manipulating color schemes, measuring distances, and determining the geometric requirements of the noncovalent interactions encountered in the protein systems.
PE was chosen as the visualization program for a variety of reasons. Among them is the ability to use the software via the web or on a local hard drive (Netscape or Internet Explorer) with the Chime plug-in. PE is also user-friendly and allows the user to utilize the Chime-based menus or command options that have been built into PE . Although PE worked very well in these assignments, any of the popular graphics programs can be used to complete the visualization assignments described in this project.
The Protein Exploration Project consisted of a series of visualization-based assignments and accounted for ∼18% of the total points in the course (equivalent to a lecture exam). The assignments were highly structured as students completed noncovalent interaction/protein structure submission forms, which contained questions and tasks related to a particular protein structure. These forms were Microsoft Word documents downloaded by students via the Course Documents folder in Blackboard. After the forms were completed, they were submitted to the instructor via the Digital Drop Box, also through Blackboard. The submission forms contained specific areas for group and protein information. Many times students were asked to identify and/or describe a potential noncovalent interaction. Therefore, the forms also had areas for students to list interacting residues or ligands and an area to describe the interaction. A description of an interaction included a discussion of the nature and type of interaction between the residues and the level of structure being stabilized [2° (secondary), 3° (tertiary), 4° (quaternary)]. The students also had to provide geometrical/distance information concerning the interaction to make a convincing argument that the two residues are in the proper position to participate in a stabilizing interaction.
Overall, these assignments focused on noncovalent interactions in proteins and probed various elements of protein structure. These assignments were designed to provide students with a certain level of three-dimensional molecular literacy, a concept described by other authors [5, 7]. The computer-based visualization methods also allow students to identify specific roles played by amino acid side chains in proteins.
Three of the assignments are described below to demonstrate the fundamentals of this approach to integrating molecular visualization into a large biochemistry course. I had the opportunity to observe and interact with many of the students in the computer lab while completing the visualization exercises. This allowed me to help the students with the assignments and to ask them questions concerning their approach to answering questions and completing tasks.
This assignment focused on ribosomal protein S6 (Protein Databank Bank ID 1LOU) . This is a small protein and works well as the first assignment because students are still learning to manipulate the structures and recognize the amino acid structures in the graphics program. There is less molecular clutter in this small protein, and students and were not overwhelmed when investigating this molecule. Below are the tasks and the question in this assignment:
List and describe four ionic interactions.
List and describe three H-bonds involving the side chain of Tyr or Gln.
Find a Ser residue forming a H-bond with a water molecule.
Interaction Question 1—
Is there a cation-π interaction between Phe97 and Lys39? If yes, describe the nature of the interaction. If not, provide details on why the two side chains are not participating in a stabilizing interaction.
To approach Task 1, the students highlighted the acidic and basic amino acids in the protein and rotated the structure to look for potential ionic interactions. Although PE will automatically highlight ionic interactions in a protein, I wanted the students to identify the basic and acidic side chains and then justify why they have the appropriate geometry to participate in a stabilizing interaction. Fig. 1 shows an ionic interaction between Glu22 and Arg82 identified by a student group. This group also correctly suggested that this interaction stabilizes the 3° structure of the protein and provided distance information to support their answer.
To successfully complete Task 2, the student groups usually highlighted acidic amino acid side chains (rich in H-bond acceptors) along with Tyr and Gln to search for potential H-bonds. Students had to apply their knowledge of the directionality and distance requirements of H-bonds (emphasized in lecture) to correctly identify legitimate H-bonds. Tasks 1 and 2 can be grading intensive because many combinations are possible. However, task 3 and interaction question 1 are easier to grade and more focused toward specific interactions. Interaction question 1 addresses π-type interactions in proteins, which play a significant role in protein folding and stability [16–18]. To answer this question, the students examined the specific amino acids and used their knowledge of the fundamentals of π-type interactions covered in lecture. With this particular question, half of the student groups indicated that there was a cation-π interaction between Phe97 and Lys39, while the other groups used geometrical information to argue that such an interaction was not significant. This provided me an opportunity to address this question in lecture and have each side discuss the geometrical requirements of noncovalent interactions. It is difficult for me to ask a similar question on a traditional lecture test or spark student discussion on the aspects of noncovalent interactions using a printed image from a textbook.
The mature bovine prion protein (residues 23–230; Protein Data Bank ID 1DWY)  was the subject of this assignment. The structure of this protein was recently solved through nuclear magnetic resonance (NMR) methods , and a .pdf file of the paper describing its structure was provided to the students via Blackboard. This paper gave the students a concise introduction to prion diseases and several references to find additional information. This paper also exposed students to an experimental method used in protein structure determination, and an example of how the structural nature of proteins is described in the literature.
This assignment was based largely on interaction questions and required the students to write a short paper on the “protein-only” hypothesis in prion diseases.
Interaction Question 1—
Is there a face-to-edge π-type interaction between Tyr145 and Tyr149? If yes, describe the nature of the interaction. If not, provide details on why the two side chains are not participating in a stabilizing interaction.
Interaction Question 2—
Is there a noncovalent interaction between Asn181 and Asp178? If yes, describe the nature of the interaction. If not, provide details on why the two side chains are not participating in a stabilizing interaction.
Interaction Question 3—
Highlight all of the Val, Met, and Ile residues in the protein. What role do these residues play in stabilizing the packing of the two large helices in the protein structure? The two long helices in this structure are not parallel to each other. Explain why this is an important structural feature observed for helices in proteins.
In at least 500 words, describe the “protein-only” hypothesis that is believed to be involved in prion diseases. You have been provided with a . pdf file of a paper entitled “NMR Structure of the Bovine Prion Protein” by Francisco López Garcia, Ralph Zahn, Roland Riek, and Kurt Wüthrich. Use this paper for background material and references.
When answering interaction question 1, most students realized that the two tyrosine side chains are perpendicular but not in the correct geometry to be involved in a face-to-edge interaction. The edge of Tyr145 is projected more toward the hydrogen atoms of the aromatic side chain of Tyr149 and not toward the body of the ring (Fig. 2).
For interaction question 2, a description of the H-bond between Asn181 and Asp178 was needed for a correct answer (Fig. 2). Having students inspect these two residues reinforced the idea that the interaction of amino acid side chains also contribute to the stability of secondary structural elements of proteins. This question also allowed me to present a situation to the students where they might be tempted to suggest an ionic interaction between the two side chains. A great deal of time in the course is devoted to explaining the ability of amino acids to participate in the various types of noncovalent interactions. Based on the lecture material, the students should understand that the side chain of Asn is an amide and will not have the opportunity to participate in an ionic interaction. Interaction questions 1 and 2 allow me to gauge the ability of students to recognize the various types of interactions involving amino acid side chains and the geometry required for a stabilizing interaction. This type of assessment can be quite difficult to achieve on a traditional lecture examination.
Interaction question 3 was also used to support structural concepts introduced in lecture. To answer this question, all the Val, Met, and Ile residues were highlighted and the two long helices in the protein were examined. To answer this question, students had to discuss the placement of these nonpolar amino acids on the helices and discuss the role of hydrophobic interactions and packing considerations in promoting the tilted arrangement of the two helices. This type of question supported the “ridges in grooves” model that was covered in class.
An important theme in the biochemistry curriculum is that “structure yields function.” Having students write a short paper concerning the molecular basis of disease (such as prion diseases) provides a relevant example to support the theme and it is also complementary to the search and examination of noncovalent interactions in proteins. The short papers also allowed me to broaden the scope of the course and provide a specific example of why it is important for students to understand the structure-function relationship in macromolecular systems.
The formation of complexes involving proteins and other types of biological molecules has implications throughout the biochemistry curriculum. The creation of visualization exercises is a way to expose students to the forces that govern biomolecular recognition and the formation of protein complexes. This particular assignment focused on a tetrameric structure of xanthine-guanine phosphoribosyltransferase (XGPRTase) complexed with guanosine monophosphate (GMP; Protein Data Bank ID 1A97) . When examining this protein structure to prepare questions concerning the interaction of amino acid side chains with bound GMP, I noticed several features of the enzyme structure that were quite intriguing. Therefore, this assignment consisted of several interaction questions that required students to apply their knowledge of structural principles to the stabilization of a multimeric protein and protein-ligand complex.
Interaction Question 1—
There are four Asp10 residues in the tetramer (chains A–D). These side chains are at the tetramer interface. Highlight bound water molecules and describe the role these Asp side chains and water molecules play in tetramer stabilization.
Interaction Question 2—
There are four Met11 residues in the tetramer (chains A–D). The side chains of these amino acids are also at the tetramer interface. Highlight these side chains (space-filling mode) and describe the role they play in tetramer stabilization.
Interaction Question 3—
Before answering this question, display the tetramer in space-filling mode. Notice how “arms” of the subunit A and subunit D (and subunits B and C) wrap around each other in an arm-in-arm arrangement. Highlight residues 140–145 in the tetramer (stick mode) and focus on the backbone atoms. Explain how the backbone atoms in these residues are helping stabilize the arm-in-arm arrangement of subunits A and D.
For the interaction questions below, highlight the bound ligand (GMP) and the indicated residue. In each question, indicate if there is a stabilizing interaction between the residue and GMP and describe the exact nature of the interaction.
Interaction Question 4—
Arg69 and GMP.
Interaction Question 5—
Lys115 and GMP.
Interaction Question 6—
Trp134 and GMP.
Interaction Question 7—
Arg37 and GMP.
Interaction questions 1 and 2 probe interactions involved in tetramer stabilization. To address question 1, students had to highlight the four Asp10 residues, which are at the tetramer interface. An inspection of just the four Asp residues may have led the students to suggest that the arrangement of these acidic residues might destabilize the tetramer through charge repulsion of the negatively charged side chains. However, when bound water molecules are highlighted, it is apparent that a H-bonding network involving water molecules and the Asp10 residues may be a factor in tetramer stabilization. Interaction question 2 asks the students about Met residues involved in tetramer stabilization. To correctly answer this question, a discussion about the nonpolar Met11 side chains interacting through dispersion forces was required. Some students suggested that the sulfur atoms of the Met residues (H-bond acceptors) were also participating in H-bonds with water molecules in the region, which seems plausible based on the placement of water molecules at the tetramer interface.
Interaction question 3 in this assignment is an example of how the visualization exercises required students to apply their knowledge of structural principles to unique situations. This question focused on the arm-in-arm arrangement that exists between two sets of dimers in the tetrameric structure of XGPRTase (Fig. 3). Once the students selected residues 140–145 of the enzyme, they used the space-filling mode to visualize the arm-in-arm structure. This question asks the students to focus only on the backbone atoms of these residues. A close inspection of this region reveals that there are H-bonding interactions between backbone atoms of subunits A and D in the region where the arms cross. Fig. 4 shows another perspective of this region and how the backbone atoms of the two subunits are in a position to form H-bonds. Most of the student groups were able to recognize the use of H-bonding interactions among backbone atoms to stabilize this dimeric unit of the tetramer, and some groups drew an analogy between the arm-in-arm arrangement in this enzyme and the formation of H-bonds in β-sheet structures.
The remaining interaction questions dealt with interactions between side chains in the nucleotide-binding region and GMP. This part of the project was assigned while nucleotide structure was being discussed in class and was designed to demonstrate the types of intermolecular interactions possible between proteins and nucleic acids. In this particular system, students were asked to recognize that the side chain of Arg69 is forming an ion-pair with the phosphate group of GMP and the ammonium moiety of Lys115 is in position to form a H-bond with the carbonyl oxygen of the guanine ring. In addition, students had to realize that the indole ring of Trp134 is in position to form a π-stacking interaction with the guanine ring of the bound nucleotide. Students need to be exposed to π interactions of this type as the literature is establishing the importance of aromatic amino acid side chains in the binding of nucleic acids [20–22]. Fig. 5 shows how these residues interact with bound GMP. Arg37, a residue in the vicinity of the GMP-bonding pocket, is also shown in this figure. Upon measuring several distances between Arg37 and GMP, most of the students decided that it is not in the correct position to form a stabilizing interaction with GMP. This type of interaction question reinforces the idea that there are strict geometry and distance requirements for the formation of noncovalent interactions, especially for ionic and H-bonding interactions.
Two recent editorials in Biochemistry and Molecular Biology Education have addressed issues associated with molecular visualization and the teaching of three-dimensional concepts [23, 24]. I believe that the visualization exercises described in this report can be placed in context with the thoughts expressed in the editorials. In her editorial, O' Leary raised some very important questions about the ability of students to deal with structural concepts in biochemistry . She also commented on the development of exercises that require students to use three-dimensional information and the use of active-learning strategies in the biochemistry curriculum. I feel that the use of computer-based molecular visualization exercises, such as the ones described in this and another report , is a step in the right direction. For example, exercises designed to probe the structural nature of proteins would not be possible without the use of computer-based graphics software that allows the user to select specific residues and to manipulate structures in three dimensions. Searching for noncovalent interactions in proteins and determining the level of structure being stabilized requires the students to think in terms of the three-dimensional nature of the structure. Finding specific interactions between proteins and ligands also forces students to think about the dimensionality of protein binding sites.
The search for noncovalent interactions in visualization projects are active-learning exercises. Students are trained to use the visualization software and often work in groups to investigate the structure of proteins and other macromolecules. The hands-on use of the software provides a student-centered approach as the students formulate their own strategy for using the software to answer the questions and complete the tasks in the visualization exercises.
In his editorial, Parslow  expressed skepticism about the use of computer-based graphics to teach structural concepts and called for more studies to assess the use of visualization techniques. He also expresses the belief that three-dimensional principles have been learned to a high standard for many years without the use of computer-based software. This may be the case, but textbooks can never provide the insight into noncovalent interactions that can be gained through the use of visualization exercises. Graphics-based assignments also allow instructors to focus on areas not emphasized in biochemistry textbooks (such as π-type interactions) and to get students actively engaged in the investigation of protein structure. On occasion, students participating in visualization exercises have observed structural motifs or interactions in which I was not familiar . This type of cooperative relationship is only possible because visualization software has been put in the hands of students. When constructing questions for assignment 3, I encountered a structural feature (arm-in-arm arrangement in XGPRTase) not seen in textbooks or when analyzing other proteins. This type of discovery fuels my desire to incorporate visualization methods into the biochemistry curriculum and is a clear example of how computer-based graphics has enriched my own understanding of protein structure.
In a recent article, Jane and David Richardson emphasized that there is little experimental data on the effectiveness of molecular graphics in teaching three-dimensional concepts and little guidance for the use of these types of materials in the biochemistry curriculum . I believe that visualization assignments like the ones described in this report can be useful for biochemistry instructors to integrate molecular visualization into both large and small biochemistry courses. Instructors can design their own exercises to emphasize a variety of issues encountered in the biochemistry curriculum that involve structural concepts.
The effectiveness of molecular visualization in enhancing three-dimensional molecular literacy has been addressed in recent publications [6–9, 25] and will no doubt remain an important issue in the molecular graphics community. I view the exercises of the Protein Exploration Project as visualization-based learning opportunities that were designed to give students a vehicle to better understand structural concepts. As addressed previously, these assignments also allow me to test the students on concepts that are difficult to tackle on traditional examinations. In fact, these assignments were not designed to enhance students' performance on lecture examinations; therefore, a controlled study that monitors test scores is not appropriate. It may be that the best assessment of these exercises is the quality of the work submitted by the students and their comments to me while completing the assignments in the computer laboratory. My informal conversations with the students took place while they were actually working on the exercises, which may be the best time to get their opinion on the assignments. A great majority of the class believed that PE was beneficial to their ability to learn about the structure of macromolecules. They also commented on how PE helps them visualize specific noncovalent interactions in proteins, which was not possible with the static images in their textbook. More than one student indicated that he/she would use PE once they were in medical/pharmacy school even if not recommended by their instructor.
The students also gave me insight into some aspects of the project that could be improved. Many of the students wanted more training in the use of PE before the assignments began and throughout the semester. Another issue with these types of assignments is the time investment of the students. Some students were concerned about the amount of time it took to complete the assignments, even though the project was helping them learn about protein structure. Based on these comments, I have decided provide continuous training throughout the semester and to limit the number of assignments to 3–4 per semester.
To date, I have been impressed with the ability of most students to learn the visualization software and to answer the questions and complete the tasks on the assignments. I am strongly committed to generating more graphics-based assignments to diversify how structural principles are taught in the biochemistry curriculum and look forward to the results of a broad-based initiative that will provide additional assessment tools to evaluate the effectiveness of molecular visualization in teaching biochemistry .
The abbreviations used are: PE, Protein Explorer; NMR, nuclear magnetic resonance; XGPRTase, xanthine-guanine phosphoribosyltransferase; GMP, guanosine monophosphate.