A Computer-Based Laboratory Experiment for ... - ACS Publications

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Biomolecules Come Alive: A Computer-Based Laboratory Experiment for Chemistry Students Naomi L. Haworth* and Lisandra L. Martin* School of Chemistry, Monash University, Clayton, Victoria 3800, Australia

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S Supporting Information *

ABSTRACT: A series of computer-based laboratory experiments have been developed to aid undergraduate students in appreciating the three-dimensional nature of chemistry and biochemistry. Both lower- and upper-division experiments have been developed. Students are introduced to the use of ChemDraw for the production of professional-quality molecular structures, Chem3D for visualizing molecules in three dimensions, and VMD for investigating protein structures and dynamics. Through a series of guided experiments, students explore important concepts in chemistry and biochemistry, such as stereochemistry, protein secondary structure, intermolecular interactions, molecular recognition, and protein dynamics. An overwhelmingly positive response to these experiments was received, with students reporting an enhanced appreciation of the nature and behavior of molecular and protein systems, and an improved understanding of lecture material. KEYWORDS: First-Year Undergraduate/General, Upper-Division Undergraduate, Laboratory Instruction, Computer-Based Learning, Drugs/Pharmaceuticals, Medicinal Chemistry, Molecular Recognition, Noncovalent Interactions, Proteins/Peptides, Stereochemistry

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is intended for lower-division (first- or second-year) undergraduates (Supporting Information, Lower Division Experiment), and the other for upper-division (third-year) students (Supporting Information, Upper Division Experiment). Both have an initial molecular structure component which involves the use of the ChemDraw/ChemOffice suite,29 followed by a two-part module in which VMD is used for the visualization and analysis of peptide/protein structures. The modules are designed to inter-relate; however, they can also easily be adapted to stand alone. In the ChemDraw module (Part I), students learn how to draw structures, change atom types and charges, and indicate stereochemistry. The resulting twodimensional structures are exported to Chem3D to produce reliable three-dimensional representations of the molecules that the students are trained to manipulate and interpret. In the second component (Part II), the VMD protein visualization package is introduced. Students learn how to use different “representations” to visualize particular aspects of protein structure, how to selectively display and investigate amino acids and protein regions of interest, and how to identify and explore enzyme binding sites. VMD is also used in Part III of each experiment, where students learn how to visualize and interpret the results of molecular dynamics (MD) simulations.

n understanding of the three-dimensional nature of molecules and biomolecules is critical for interpreting their reactivity and function. Often, however, in undergraduate teaching (and even in scientific research), only two-dimensional depictions of the systems of interest are considered. The value of using molecular modeling software to aid undergraduate students in visualizing molecular and biochemical systems is being increasingly recognized.1−8 Various workshop exercises have been developed using commercial software,3,9−17 as well as self-designed computer programs;18 these have generally been well-received by students. However, the use of these specialist packages requires the purchase of software licenses, meaning that the exercises cannot be adopted by departments experiencing budgetary constraints. In terms of free software, Rasmol19,20 was the package of choice in the early 2000s for introducing undergraduate students to biomolecule visualization.2,11,21 More recently, however, its use in the research community has been superseded by programs such as Pymol22 and VMD (Visual Molecular Dynamics).23,24 While several exercises that employ these state-of-the-art packages have been designed for advanced biochemistry and molecular biology courses,25−28 resources suitable for introducing molecular and biomolecular visualization to chemistry students are lacking. In this paper, two computer-based exercises are reported. These are designed to be used as “dry-lab” experiments within undergraduate chemistry laboratory courses. One experiment © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: December 5, 2017 Revised: September 13, 2018

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DOI: 10.1021/acs.jchemed.7b00931 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. ChemDraw and Chem3D screenshots showing the following: (A) D- and L-alanine are mirror images of each other (top) and are nonsuperimposible (bottom); (B) the 6-membered ring of phenylalanine is planar, while the ring in glucose is puckered.

group problem solving were encouraged. Assessment primarily involved a written report detailing the answers to the questions posed throughout the experiment. Learning by the upperdivision students was also assessed in a midsemester test. Note: There are no hazards associated with this computational laboratory exercise.

Three educational objectives were targeted in the development of these experiments: 1. To improve students’ appreciation of 3D concepts in chemistry (e.g., stereochemistry, planarity, conformational flexibility) and the relationship between 2D and 3D representations of molecules. 2. To improve students’ appreciation of the 3D nature of biomolecules. 3. To help students understand that biomolecules are not static but can change shape dramatically in response to external events. An additional benefit of using ChemDraw/Chem3D and VMD in the teaching laboratory is that both packages are widely used in both research and industry. The experiments were therefore also designed to train students in important computer-based skills required for future careers in the fields of chemistry and structural biology.

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DISCUSSION

Part I. Understanding Molecules in 3D: ChemDraw and Chem3D

In the lower-division molecular structure module (Part I), ChemDraw and Chem3D are used to explore basic chemical concepts such as chirality and planarity. Students produce screenshots illustrating how D- and L-amino acids are “nonsuperimposible mirror images” of each other, and showing that although 2D structures might lead one to think that the 6membered rings of phenylalanine and glucose are equivalent, in reality one is planar and the other puckered (Figure 1). This module was performed successfully by almost all students. Of those who attempted to produce an image showing that the molecules were mirror images, 90% did so correctly, while 95% of students who included images to show that the two isomers were nonsuperimposible were successful. (Note: 22% of students only produced one set of images.) Eighty percent also recognized the planarity in phenylalanine and nonplanarity of glucose and correctly attributed this to the hybridization of the carbon atoms. These good results indicate that this computer-based approach could provide a valuable tool for introducing the traditionally challenging concept of chirality. Material in Part I for the upper-division students was intended to briefly revise ChemDraw use, before moving on to the drawing and visualization of the complex biomolecules and peptides to be investigated later in the experiment. However, it transpired that only 20% of the student cohort in the introductory year acknowledged any previous exposure to ChemDraw and only 37% reported having used any molecular graphics software at all before commencing the course (assessed using the Upper-Division Workshop Pre-Exercise Questionnaire provided in the Supporting Information). It was therefore necessary to allow students more time than planned to complete this module. This did, however, result in students demonstrating excellent mastery of ChemDraw skills in their

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EXPERIMENTAL SECTION Each experiment was designed to be completed during a single laboratory session in an on-site computer lab as part of a regular laboratory program. The lower-division experiment is intended to be completed in 3 h, and the upper-division version is designed for a 4 h session. In our implementation, the upper-division experiment forms part of a final-year undergraduate Medicinal Chemistry course. The lowerdivision experiment was initially designed for a first-year Chemical Biotechnology subject and was later redeployed in a second-year Biological Chemistry laboratory program. All students enrolled in the relevant course completed the experiment during the same week; this involved several parallel sessions, with each class comprising no more than 30 students. The same demonstrators presented all sessions; in general, this included a senior faculty member and three graduate students or postdoctoral researchers as assistants. Access to ChemOffice was provided to all students via a University-wide site license. The lower- and upper-division experiments have now been presented on four and five occasions, respectively. In this report, data are presented for the first year of implementation in which the lower-division cohort was 49 and the upperdivision cohort 59. Subsequently, these experiments were used in courses with enrollments of up to 161. For both the upperand lower-division experiments, students worked independently and produced individual reports; however, discussion and B




Figure 2. VMD screenshots showing the structures of (A) α-conotoxin Vc1.1 and (B) Acinetobacter baumannii transpeptidase.

Figure 3. Results from student reports showing (A) identification of residue−residue interactions ([Residue 1 no.]−[Residue 2 no.] [Interaction type]) in conotoxin by lower-division cohort (49 students) and (B) identification of the residue−residue and residue−inhibitor interactions in transpeptidase by upper-division students (Qn.n indicates the relevant question in the worksheet). Note that not all the upper-division cohort attempted Q2.11; cases where no answers were given are excluded from the analysis, reducing the cohort size from 59 to 38 for this question. Colors are used to highlight the different interaction types; pale colors indicate successful identification of an interaction but incorrect labeling.

hydrophobic interactions (Figure 2A). A more complex protein, Acinetobacter baumannii transpeptidase, was chosen for the upper-division experiment. This contains a wide variety of secondary structure elements, including α and 310 helices, parallel and antiparallel β sheets, β bridges, β turns and unstructured loop regions, disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions (Figure 2B). A second advantage of this system is that X-ray crystal structures are available for transpeptidase both in its apo form (PDB: 3udf)30 and when bound to its inhibitor, penicillin (PDB: 3udi).30 This gives the students the opportunity to investigate the binding site of the enzyme and the interactions which give rise to inhibition. For this module, truncated versions of the two PDB files are provided, containing only the catalytic domains. After an initial exploration of the secondary structure elements of their respective biomolecules, students in both divisions move on to the central task: investigating inter- and intramolecular interactions. Lower-division students are given a list of residue pairs and asked to characterize the interactions they are engaging in. Upper-division students are given a more open-ended task in which they are given a range of residues in the sequence and asked to identify all interactions of a certain type (Q2.5−2.7). Subsequently, the upper-division students are also asked to identify which residues form part of the

reports, with the average grade for the relevant questions being 94%. Nevertheless, it might be hoped that, by the final year of their undergraduate studies, students would be routinely using molecular graphics packages to produce professional molecular structures for laboratory reports. In this cohort, only 18% reported having done so prior to this experiment, with the remainder simply drawing structures by hand. Several weeks after this session, usage of ChemDraw structures in laboratory reports was assessed, unannounced. While the percentage uptake had almost doubled, incidence was still well below 50% (37% of reports). This highlights the importance of introducing these skills early in an undergraduate program. As a result, in subsequent years the ChemDraw module of the lower-division experiment was introduced into the core second-year curriculum. Part II. Visualization and Interpretation of Protein Structures: VMD

Once the molecular structure module is complete, students move on to investigating the 3D structures of peptides and proteins (Part II). The lower-division students study a small peptide, α-conotoxin Vc1.1, which contains a variety of structural elements, including an α-helix, disulfide bridges, and a salt bridge, in addition to hydrogen bonding and C




Figure 4. (A) A selection of the structures given to the 59 upper-division students as part of their midsemester test. (B) The marks achieved by students for selected test questions.

Part III. Protein Flexibility and Conformational Change: VMD

penicillin binding site and the nature of the interaction(s) of each residue with the inhibitor (Q2.10−2.11). The students’ success in identifying these interactions is illustrated in Figure 3. Hydrogen bonds (blue), electrostatic interactions (yellow), and covalent bonds (including peptide bonds and disulfide bonds, as well as the covalent bond between transpeptidase and penicillin; red) were recognized by almost all students. Incomplete marks for Q2.5−2.7 of the upper-division experiment generally resulted from the student missing one of several interactions present. There were some issues with labeling, however. Significant percentages of students (24% of lower-division and 50% of upper-division) failed to recognize the electrostatic character of interactions between charged residues, identifying these as simply hydrogen bonds (a technically correct but incomplete response). In later years, the instructions were expanded to highlight the need to pay particular attention to whether electrostatic interactions are present. In the upper-division module, students also struggled to correctly identify the covalent bond between residue Ser434 and penicillin. The instructions were also updated to change the way the students were instructed to view the system to make this interaction clearer. The identification of hydrophobic interactions is inherently more challenging than other interaction types; this was reflected in the student reports, where these interactions were only successfully identified in ∼50% of cases (green in Figure 3). It is important to note that, in the lower-division study, each pair of residues with hydrophobic interactions also engaged in a second interaction type. Many students only gave a single interaction type for each residue pair, which may have biased the results. Hence, overall both lower- and upper-division cohorts demonstrated successful identification of the interactions responsible for protein structure and inhibitor binding in their systems.

The experiments for both divisions conclude with an analysis of a short molecular dynamics simulations of a small peptide (Part III). Lower-division students continue with their study of α-conotoxin, exploring the high stability of the overall fold, the movements of flexible residues, and the effects of cleavage of the disulfide bonds on the peptide structure. For the upperdivision students, a molecular dynamics simulation of the hormone oxytocin is provided. They are also asked to identify flexible residues within the structure as well as the intermolecular interactions which are important in different conformations of the hormone. Unfortunately, due to the extended time required by the upper-division students for the ChemDraw module in its introductory year, a significant proportion of the cohort was unable to complete this module. Hence, only the results for the lower-division students have been assessed. These students performed extremely well in this module: 90% correctly reported that the reduction of the disulfide bonds of conotoxin increased the flexibility of the system, and 98% recognized that these bonds were important for structural stability. In addition, 93% were able to identify the most flexible of the conotoxin side chains, and a further 77% also recognized that it only formed weak interactions with the remainder of the peptide. In subsequent years, the upper-division students had no difficulty completing the experiment in the allotted time. This allowed the introduction of an oral assessment question (Part IV). A choice of two exercises was provided, in which students explore proteins that undergo large conformational changes as part of their function: calmodulin (PDB: 1cfd, 1ckk, 1cm1),31 a critical signaling protein; or CLIC1, which alternates between a membrane-bound ion channel (PDB: 1rk4) and an enzymelike cytoplasmic structure (PDB: 1k0m) with the oxidation or D




Figure 5. Student response to the experiments. (A) Student evaluation of the value of the experiments in terms of how interesting they found them, how confident they were in performing them, and the skills and understanding they believed they had gained. (B) Student feedback on their favorite parts of the experiments. Lower-division cohort = 49 students. Upper-division cohort = 59 students.

reduction of a disulfide bond.32 Students were asked to load and display these structures and to answer questions on these systems posed by a demonstrator.

positive, as shown in Figure 5A, with the exercises being rated well above average in terms of how interesting the students found them, how confident they were in performing the tasks, and how they believed the experiments enhanced their scientific skills and understanding. Students were also asked which parts of the experiments they found most enjoyable; a fairly even split between all modules was reported for both upper- and lower-divisions (Figure 5B). The only section that was less favored was the simulation section of the upper-division experiment. This was almost certainly due to an appreciable proportion of the students not attempting this final module in the year this feedback was sought. A selection of student responses to these experiments are provided in the Supporting Information.

Assessment of Student Learning: Interpretation of Protein Structures

To further assess whether the concepts of protein structure and residue−residue interactions were successfully retained by upper-division students, a question involving protein structure interpretation was included in their assessment (Supporting Information, Upper Division Workshop Assessment). Students were given a series of images, including both ChemDraw and VMD screenshots of a short β hairpin peptide, and asked to identify various structural elements (see Figure 4). Generally, students performed well in this assessment. Structural elements such as termini and covalent interactions were correctly identified by most students (88% and 90%, respectively). Again, students found the correct identification of electrostatic and hydrophobic interactions more challenging; however, success rates were similar to those in the laboratory experiment (52% and 44%, respectively).

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CONCLUSION AND FURTHER DEVELOPMENT Two computer-based laboratory experiments have been developed to aid undergraduate students in appreciating the three-dimensional, dynamic nature of chemistry and biochemistry. Student feedback indicated that these educational objectives were successfully achieved. Experiments are provided at both introductory and more advanced levels, allowing this material to be employed for first-, second-, or third-year undergraduate education. The modular nature of the experiments also allows the molecular structure (ChemDraw/

Student Response to the Experiments

The responses of the students to both experiments (assessed using Supporting Information, Upper Division Workshop Student Feedback Questionnaire) were overwhelmingly E




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Chem3D) and biomolecular analysis (VMD) experiments to be presented separately if desired. Given that the vast majority of students nowadays are confident with a three-dimensional interpretation of virtual environments, molecular visualization software, such as Chem3D, also provides a valuable tool for the teaching of fundamental but challenging concepts such as chirality. Indeed, the molecular structure module of the lower-division experiment has been co-opted into the core second-year undergraduate curriculum as an online assessment exercise to both introduce vital ChemDraw skills and reinforce important firstyear concepts. The exercise has been slightly modified so that complementary material is presented to students who take both the core and Biological Chemistry courses. In the Supporting Information, three separate workshops have therefore been presented: the original lower- and upperdivision experiments, as well as the stand-alone lower-division molecular structure exercise. Assessment tools are also included. Educators are invited to contact the authors for access to sample answers to the student assessment questions.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00931.

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Student handouts for lower- and upper-division experiments plus the lower-division ChemDraw/Chem3D exercise; protein structure and molecular dynamics files; student questionnaires; and demonstrator notes and marking schemes (ZIP)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Naomi L. Haworth: 0000-0002-3299-3137 Lisandra L. Martin: 0000-0003-0486-5813 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge funding from the Monash University Faculty of Science Education Projects Grant Scheme (to L.L.M.). They also thank Irina Simonova and Mingdeng Luo for assistance in the development and testing of the experiments and Erica Haworth for data handling and analysis.

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A Computer-Based Laboratory Experiment for ... - ACS Publications

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