Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Q 2010 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 38, No. 6, pp. 359–364, 2010 Article Beyond Textbook Illustrations: Hand-held Models of Ordered DNA and Protein Structures as 3D Supplements to Enhance Student Learning of Helical Biopolymers Received for publication, April 9, 2010, and in revised form, May 11, 2010 Karnyupha Jittivadhna, Pintip Ruenwongsa, and Bhinyo Panijpan‡ From the Institute for Innovative Learning, Mahidol University, 999 Phuttamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand Textbook illustrations of 3D biopolymers on printed paper, regardless of how detailed and colorful, suffer from its two-dimensionality. For beginners, computer screen display of skeletal models of biopolymers and their animation usually does not provide the at-a-glance 3D perception and details, which can be done by good hand-held models. Here, we report a study on how our students learned more from using our ordered DNA and protein models assembled from colored computer-printouts on transparency film sheets that have useful structural details. Our models (reported in BAMBED 2009), having certain distinguished features, helped our students to grasp various aspects of these biopolymers that they usually find difficult. Quantitative and qualitative learning data from this study are reported. Keywords: Helical structure, DNA, polypeptide, science education. Helical structures of biopolymers are ordered and have symmetry in three dimensions. College students have to learn about helices of the double-stranded DNA, the single-stranded protein a-helix, and possibly others in bioscience classes. Even high school students studying biology are exposed to these common helical structures and are examined for their knowledge of these structures. However, when asked to draw a sketch of the DNA double helix with as little details as that drawn by Francis Crick’s wife, Odile, for the seminal Nature paper in 1953 [1], quite a few students, including some graduates, cannot do it well: no clear helical (right-handed) sense, haphazard hydrogen bondings between basepairs, no major and minor grooves, the strands are not antiparallel, and so forth. Students do not perform much better with the bare-backbone polypeptide a-helix: no clear helical (right-handed) sense, haphazard hydrogen bondings, each amino acid residue is not quite correct. Given these difficulties in student learning this aspect of chemical biology, researchers recommended enhancing the learning of DNA and protein structures through the use of three-dimensional physical models [2–4]. Herman et al. [5] and Bain [6] reported that concrete visual models that can be easily manipulated, play an important role in capturing the interest of students at both high school and undergraduate levels and encouraging more ‡ To whom correspondence should be addressed. Tel.: þ66 (0) 2441-9020; Fax: þ66 (0) 2441-0479. E-mail: [email protected]. This work is supported by Tertiary Education Commission, Ministry of Education. This paper is available on line at http://www.bambed.org sophisticated thinking about the tangible molecular world. It should be noted that one obvious limitation to the widespread use of physical models is their high cost and limited availability. To provide our students with an easy access to a useful learning tool, we had developed physical models of ordered structures of the B-form DNA, protein a-helix, and b-pleated sheet made from computer-printed transparency film cut-outs. We used the already-assembled DNA double helix with two central axes (made from straight wires) [7] to teach students, medical as well as graduate bioscience, to learn the essential structural details of DNA and their role when interacting with drugs and proteins using the hydrogen bond donor and acceptor groups on the bases and the electrostatic charge on the phosphate group. The relative spacings of the major and minor grooves change with the tilting of the base pairs from B to A form and from right-handed to the left-handed conformation, simply by manipulating the two central axes, illustrate their conformational change without breaking any covalent bond. Because the printed base pairs are transparent, the stacking of successive base pairs and their relative twist (of the stacking) can be seen and measured. Our polypeptide a-helix and b-pleated sheet (parallel, antiparallel) with connecting polypeptide turns (having only alanine side chains for simplicity) show their regular backbone and regular side chain arrangement, a feature students usually miss. The instruction for building these parsimoniously designed models is described in a recent article in this journal [7]. The model enables the instructor to simplify, illustrate, and explore these chemical features 359 DOI 10.1002/bmb.20427 360 BAMBED, Vol. 38, No. 6, pp. 359–364, 2010 as they are made to scale and provide atomic details with the conventional CPK color scheme. The aims of this study: (1) to identify the areas where students have difficulties in understanding these biologically important helical structures they are exposed to only as illustrations in textbooks and (2) to assess the contribution of our models to students’ understanding of ordered DNA and protein structures and their functions. All these were based on both quantitative and qualitative data from educational studies. STUDY DESIGN AND METHODS Because the polypeptide chain, the protein a-helix and b-sheet, the polynucleotide chain, and the B-form DNA are common biochemical structures taught in nearly every introductory biology courses, we were thus able to conduct this educational study at multiple sites and with students of various academic backgrounds. The study was done during the year 2006–2009 with 498 student volunteers: 9th–12th graders (aged 14–19) in high schools, freshmen and sophomores, and preclinical medical students (aged 17–20), who had previously been exposed only to textbook presentations of DNA and protein structures during their earlier school/academic years. Three instruments were used in this study: a multiplechoice content questionnaire, an open-ended content questionnaire, and another open-ended questionnaire, which was used for group interviews. All questions were specifically developed for this study. The multiple-choice and open-ended content questionnaires were administered as the pretest (before modelhandling activity but with easy access to textbooks) and posttest (during the model-handling activity), both containing the same questions. The students received the open-ended written questionnaire after they handed in their responses to the multiple-choice questionnaire. The group interviews were conducted at the end of the activity session. The multiple-choice questionnaire, which included nine questions, was filled out by all the 498 students. These were questions on base pairing, hydrogen bond donoracceptor possibility of each of the bases, polynucleotide strand polarity, general equivalence of both ends of the double helix, location of grooves with respect to the base pairs and the backbones, configuration of the peptide bond, number of hydrogen bonds in an a-helix spanning 12 amino acid residues, the 21 helicity of the bpleated sheet, and the possibility of the reverse turn in the sheet. Each student answered the questions individually, but they were allowed to discuss the material as groups of four to six. During the pretest, students were also allowed to consult the textbooks provided for them. The posttest survey was carried out while the students were handling the models to exploit the latter’s educational potential. Students in each group were allowed to manipulate the complete set of models and some components, especially the base pairs of the DNA modeling kit, and participated in reasoned discussions about some structural features of the models following the main issues guided by the test questions. Student learning gain was quantified as a percent of students in the trial, who changed their answers from incorrect at pretest to correct answer at posttest for each item. The open-ended written questionnaire included five questions and was given to 28 volunteer student groups after they completed the multiple-choice questionnaire. This evaluation process required some integration of the details of the factual knowledge on DNA and protein structures, which included in the multiple-choice questionnaire (1) Would Watson–Crick base pairs of different orders, for example, A:T versus T:A, G:C versus C:G affect the regularity of the sugar-phosphate backbone? Explain your answer, (2) Draw the two complementary DNA chains showing in detail the two deoxyribose-phosphate backbones, (3) How can you describe the nature of the handedness of the B-form double helix, and its grooves?, and (4) Draw a schematic diagram showing a repeat of a helix with the number (n) of residues ¼ 4 and 2. In evaluating the open-ended questions, we looked for the accuracy of their descriptive responses and the number of student groups that could not answer the questions descriptively at all. In group interviews conducted by the first author after the content questions, students were asked to answer two additional questions, which directed them to talk about the contribution of the physical models to their learning (‘‘Do you feel more confident completing the questions on DNA and polypeptides while handling this set of models?’’ And ‘‘How different types of representation (e.g., textbook pictures, computer illustrations, other physical models, and this set of models), used as enabling tools, help facilitate your learning of the molecular structures?’’). RESULTS AND DISCUSSION The aim of this work was two-fold: (1) obtain quantitative data in an effort to link student test performance with the use of the physical macromolecular models in terms of structures and (2) obtain qualitative data to corroborate the quantitative data and to define the areas where students have difficulty in understanding the material. Factual Knowledge Students’ factual knowledge was evaluated by means of multiple choice questionnaire, which has nine questions on both DNA and the polypeptides. Questions and responses to the pretest and posttest can be found in Table I. The findings from the pre- and posttest survey showed that in the pretest, only questions 1 and 5 were answered correctly by more than 50% of the students, whereas, in the posttest, the vast majority of them had the correct answers for all nine questions. The percentage difference in correct answers of postversus pretest for each question was also large: 26.7, 64.1, 42.2, 64.9, 42.2, 59.2, 68.7, 90.8, and 49.0 percent for questions 1–9, respectively. Analysis of students’ answers to the open-ended questionnaire showed that similar to the findings from the multiple-choice questionnaire, student performance was significantly better on the pretest containing the 361 TABLE I Questions and responses to the pretest and posttest multiple-choice questionnaire Pretest Questions n 1. Which nitrogenous base pair is present in the same proportion in double-stranded DNA molecules? 1. Cytosine; Thymine 18 365 2. Thymine; Adeninea 3. Adenine; Guanine 27 4. Guanine; Cytosine; Adenine; Thymine 88 Posttest % n 3.6 73.3 5.4 17.7 0 498 0 0 % 0 100 0 0 2. Consider the Watson–Crick base pair below; there are the hydrogen bond donors and hydrogen bond acceptors. When accessing the bases from the major groove what does one encounter—going from left to right? 1. 2. 3. 4. Acceptor-donor-acceptor Acceptor-acceptor-donor Donor-acceptor-acceptora Donor-acceptor-donor 269 32 136 61 54.0 6.4 27.3 12.3 9 2 455 32 18.1 0.4 91.4 6.4 3. Knowing that a glycosidic bond links the sugar to a base what is true for a Watson–Crick base pair in a duplex B-DNA molecule? 206 41.4 416 83.5 1. The beta-glycosidic bonds point in the opposite directionsa 2. The alpha-glycosidic bonds point in the opposite directions 222 44.6 82 16.5 3. The beta-glycosidic bonds point in the same directions 38 7.6 0 0 4. The alpha-glycosidic bonds point in the same directions 32 6.4 0 0 4. What will result from a 1808 flip of the principal axis of B-DNA structure? 1. Left-handed B-DNA 2. Right-handed B-DNAa 3. Left-handed Z-DNA 4. Right-handed A-DNA 268 155 57 18 53.8 31.1 11.5 3.6 2 478 3 15 5. Where is the principal helix axis of the ideal B-DNA located? 1. Through major groove 2. Through minor groove 3. Through the middle of the Watson–Crick base pairsa 4. Through P atoms in the backbones 121 77 288 12 24.3 15.5 57.8 2.4 0 0 498 0 6. The peptide bond in a perfect protein a-helix: 1. is planar. 2. is trans in configuration. 3. is rotatable about the single bonds linked to the a-carbon 4. Options 1, 2, and 3 are all correcta 85 308 62 43 17.1 61.9 12.5 8.6 132 21 7 338 26.5 4.2 1.4 67.9 7. In a typical a-helix of only 12 residues: 1. there are eight H-bonds. 2. the first four amide hydrogens do not participate in the helix H-bond. 3. the last four carbonyl oxygens do not participate in the helix H-bond. 4. Options 1, 2, and 3 are all correcta 213 87 166 32 42.8 17.5 33.3 6.4 9 17 98 374 1.8 3.4 19.7 75.1 8. The protein b-pleated sheet structure: 1. is pleated because the peptide bond is not planar 2. has the side chains of adjacent amino acids only on one side of the plane of the sheet 3. has the backbone which can be regarded as a helix with two residues per turna 4. makes a zigzag pattern perpendicular the chain directions 52 243 38 165 10.4 48.8 7.6 33.1 6 0 490 2 1.2 0 98.4 0.4 234 127 78 59 47.0 25.5 15.7 11.9 478 6 10 4 96.0 1.2 2.0 0.8 9. Which statement is true about the b-sheet backbone connection? 1. The shorter b-turn allows the sheet to reverse the direction of its polypeptide chaina 2. The longer crossover connection connects the antiparallel strands of the sheet 3. The H-bonds formed in the crossover connection structure are bent significantly 4. One or more b-strands may lie between the two strands connected by the crossover connection but the latter never links the two nearest neighboring strands n, no. of responses. a Correct answer. 0.4 96.0 0.6 3.0 0 0 100 0 362 BAMBED, Vol. 38, No. 6, pp. 359–364, 2010 TABLE II Responses to the pretest and posttest open-ended content questionnaire Pretest (n ¼ 28) Questions 1. Would Watson–Crick base pairs of different orders, for example, A:T versus T:A, G:C versus C:G affect the regularity of the sugar-phosphate backbone? Explain your answer. 2. Draw the two complementary DNA chains showing in detail the two deoxyribose-phosphate backbones. 3. How can you describe the nature of the handedness of the B-form double helix, and its grooves? 4. Draw a schematic diagram showing a repeat of a helix with the number (n) of residues ¼ 4 and 2 Posttest (n ¼ 28) C U ND NA C U ND NA 3 2 22 5 0 1 8 15 4 21 1 2 21 6 0 1 2 17 6 3 23 3 1 1 2 23 0 3 25 2 0 1 n, no. of student groups; C, correct; U, unclear; ND, no description, NA, no answer. same questions. The wider variability between students’ responses to open-ended questions was due to the level of accuracy of their descriptive responses. There were fewer student groups that did not answer some or all of the questions. The findings from the pretest open-ended questions shown in Table II indicated that some students were not even aware of the following: 1. A nucleotide in a double helix is related to the nucleotide lying across the helical axis from it in the same plane by a pseudo two-fold axis of rotation and that the two types of Watson–Crick base pair have exactly the same geometry. 2. When looked at end on, the relative rotation angle between one base pair and the pair away from the onlooker is 368 clockwise although students can verbalize that one turn of the helix encompasses 10 base-pairs. 3. Each phosphate group makes two ester linkages: one with the oxygen of C30 of the deoxyribose, and one with the oxygen of C50 . 4. C10 of the sugar is covalently linked to the nitrogen 9 of a purine or the nitrogen 1 of a pyrimidine. 5. In the major groove, a large portion of nitrogenous bases are exposed or more accessible. 6. Definition of a helix, especially a helix with two residues per turn. Conceptual Learning Results from the two assessment methods showed that from their exposure to our models, even for a relatively short time, students gained relevant conceptual knowledge of biomolecular structures. This is illustrated by the following examples of student quotations. ‘‘Only Watson–Crick A:T, T:A, G:C, and C:G pairs allow the construction of a regular double helix. This is because of a size complementarity, the large bases always pair with the small bases. We also noticed when handling the base pair unit pieces of the DNA modeling kit that the two types of base pair also have exactly the same geometry, that is, AþT and CþG span virtually an equal distance and space between the sugar-phosphate backbones. Thus, assorted Watson–Crick pairings of the bases do not perturb the arrangement of the sugars’’ (Response to item 1). ‘‘I understand better now the directionality of the DNA chains after working with the physical model. The backbone has directionality because the phosphate group is linked via oxygen to the 30 carbon atom of one sugar and to the 50 carbon atom of the other’’ (Response to item 2). Naturally, the two ends of a perfect DNA/RNA double helix are identical in that both have the 50 and 30 ends and there is at least a pseudo two-fold axis of symmetry relationship between them: when looked at end-on both helices are right-handed. For the a-helix, although it is right-handed going from the N to C or C to N terminals, the N and C terminals are different, and in going from C to N and N to C one encounters things in opposite orders. The same applies to the two different ends of each strand of the nucleic acid double helix. Another difficulty students have is in telling the 50 to 30 direction for DNA and the N to C direction for protein from inside the chain, that is, not from the ends. For DNA and RNA, they have to learn that C10 is bonded to the base and C50 is outside the ‘‘plane’’ of the ring, thus C30 can be identified more easily. The instructor may make it more memorable (with no extra basic structural learning) by saying that as a mnemonic: one goes up or down the staircase of the major groove’s base pairs with one’s right hand holding onto the 50 to 30 balustrade. For polypeptides, it goes as N Ca CO and this is easy to learn. ‘‘The model of protein a-helix with the guiding arrow helps indicate the handedness and the direction of the polypeptide backbone more clearly. When the instructor asked me to look at each one end-on I found that when the turn is clockwise then the helix is right-handed and when it is anticlockwise, the helix sense is left-handed’’ (Response to item 3). ‘‘It is much easier than looking at the 2D illustrations and seeing the chains turn to the right as they move sideways, upward or downward’’ (Response to item 3). The students were asked to look at each model endon and describe the helical turn further and further away from the eye of the onlooker. When the turn is clockwise then the helix is right-handed and when it is anticlockwise, the helix sense is left-handed. We have found this to be much more efficacious in the long run than using the left-handed or right-handed rule borrowed from physics classes. One caveat, nowadays, with digital 363 watches and clocks, some students do not know which is clockwise or anticlockwise. ‘‘The stacking of base pairs upon each other creates a helix with two grooves because the single bonds formed C10 of the two deoxyriboses, with the base pairs, on the ‘‘same’’ plane, make an angle lower than 1808, the grooves are thus unequal in size’’ (Response to item 3). ‘‘I have learned about the major and minor groove before, but I did not realize until now that each base has a pattern of hydrogen bond acceptors and donors, which, when the bases are stacked on top of one another can offer a sequence that interacts with the complementing amino acid side chains of an interacting protein’’ (Response to item 3). The differences between the major and the minor grooves of the B-form double-stranded DNA cannot be appreciated unless one scrutinizes a good model. In our experience, a hand-held skeletal model is superior to a computer-rendered one, even when the latter is rotatable on the screen. Apart from the obvious differences in groove size between the two polynucleotide backbones across the major and the minor grooves, reaching the outer edges of the base pairs from the major groove side is easier than doing it from the minor groove side, which has different parts of the bases partially blocked by the sugars. The case for 3.6 residues per turn a-helical polypeptide deriving from 18 residues per 5 turns can be easily made, whereas 3.6 residues per turn is not very convincing to students looking at a helix with only a few turns. Six-tenths of a turn is difficult to arrive at. Also the helical arrangement of the amino acid side chains can be easily seen. Even in the case when the helix is not perfect one can see how the amino acid side chains of a helical polypeptide can interact (more or less helically) with side chains of another polypeptide chain, and/or the polypeptide backbone. The side chains can also interact with the base-pairs of DNA or RNA or even the sugar-phosphate backbone. ‘‘I have learned from the b-pleated sheet model of polypeptide that for n ¼ 2, the helix degenerates into a nonchiral backbone’’ (Response to item 4). That the b-pleated sheet can be considered as a helix so stretched that most backbone atoms are more or less in the same plane could be made clearer to the students with the hand-held models. Student Model Building Before we came up with the computer-printout models as reported earlier [7], we used to ask groups of graduate students (4–5 per group) to make their own DNA (A and B forms) or RNA (A-form) double helix from transparency film sheets on which the base pairs and the sugar-phosphate backbone were drawn by the students. All groups succeeded in correctly working out the proportion of the distance between successive base pairs, helical diameter and helical repeat, so forth. Nevertheless, they reported having problems with the proper folding of two sets of double-slanted parallel lines drawn on a sheet which, when folded properly would give the right-handed double helix with a major and a minor groove. If folded the other way, the helical sense would be wrong and the Watson–Crick base-pairs would not link with the corresponding C10 of sugars. For high school students, we usually give them Watson–Crick base pair print-outs on the transparent plastic top of the sweet container. These flat tops, each with a hole in the middle, are then arranged with flat surface perpendicular to and along a drinking straw (acting as the central axis) with pins for regularly separating the base pairs. The backbone was given as a sheet of transparency with two sets of double-slanted parallel lines (with sugar-phosphate structures as the backbone drawn on them). Students are asked to fold this sheet of transparency to give a right-handed double helix. Again, they have to learn how to fold the sheet properly as well as to rotate the base pairs one after the other at a correct angle (actually determined by two lines marked on the transparency tops) relative to each other. Otherwise, the backbone and the base pairs would not match. Here also students learn a great deal by construction. Although graphical models are relatively easy to find on the internet, few of them offer clear information and explanation about their physicochemical characteristics and other contextual information for the needs of the inexperienced beginning students [8, 9]. Thus, concrete 3D models should still be made available to students at least in the introductory class to initially anchor the basic understanding. A transition to the use of virtual computer models can be made later on for better interactivity and more precise bonding and structure values. CONCLUSIONS Utility of Models The attitudinal data obtained provided clear evidence that a majority of students reacted positively to the models and believed that their understanding improved when handling this set of models. Most students elaborated on the difficulties of understanding the abstract image of structural diagrams found in textbooks. Those who had additional experience in using molecular visualization software stated that most freeware downloads, especially, the space-filling ones, did not provide much assistance to the inexperienced users, as found by others [8, 9]. Some students found that certain popular physical models they had used could not give them an insight into the atomic details. Our models offer very tangible structural details that students could learn by themselves or only with a little guidance from the instructor. Features of the DNA double helix, such as helical sense, base stacking, adjacent base pairs’ relative rotation, major and minor grooves, accessibility to hydrogen bond donors and acceptors, backbone direction (50 to 30 ), and so forth, can be grasped more easily. We wish to elaborate more on accessibility to the major and minor grooves. Apart from the obvious differences in the two widths of the B-form, the charged phosphate groups, and the hydrogen bond donor and acceptor parts of the various bases are the extras the students can see easily and be used for their learning of 364 DNA interactions with groove-oriented drugs and protein chains. It is worth pointing out that these grooves can have one pseudo two-fold axis of symmetry, which can selectively interact with molecules having a similar symmetry. Naturally, intercalation by drugs and dyes can be easily visualized because of the gap between base pairs. However, distortion of the helical axis and base pairs because of intercalation is not as dramatic as in the space-filling models. Our transparency-based computer printout models are superior in many ways, for example, easily constructed while having atomic details that can be looked at through many base pairs or residues from both ends, and they are also portable and robust. One possible drawback is that the base pair thickness is not shown as can be seen in some space-filling models, textbook, and computerbased illustrations. But the instructor can inform the students of the drawback and make corrective statements. For a-helix and b-pleated sheet models, students can also follow easily the backbone direction, adjacent amino acids side chain relative positions, helical sense, etc. The instructor may be reminded that in many proteins, the helical and sheet parts may not be so perfect and the axes can fold and wrap around other biomolecules. As a result of using our models not only could students answer the multiple choice questions better at posttest but also describe verbally what they know more cor- BAMBED, Vol. 38, No. 6, pp. 359–364, 2010 rectly, and at length, and with relatively fewer groups unable to answer any question at all. REFERENCES [1] J. D. Watson, F. H. C. Crick (1953) Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid, Nature 171, 737–738. [2] L. M. Beltramini, A. P. U. Araújo, T. H. G. de Oliveira, L. D. dos Santos Abel, A. R. da Silva, N. F. dos Santos (2006) A new three-dimensional educational model kit for building DNA and RNA molecules: Development and evaluation, Biochem. Mol. Biol. Educ. 34, 187–193. [3] M. A. Harris, R. F. Peck, S. Colton, J. Morris, E. C. Neto, J. Kallio (2009) A combination of hand-held models and computer imaging programs helps students answer oral questions about molecular structure and function: A controlled investigation of student learning, CBE Life Sci. Educ. 8, 29–43. [4] J. R. Roberts, E. Hagedorn, P. Dillenburg, M. Patrick, T. Herman (2005) Physical models enhance molecular three-dimensional literacy in an introductory biochemistry course, Biochem. Mol. Biol. Educ. 33, 105–110. [5] T. Herman, J. Morris, S. Colton, A. Batiza, M. Patrick, M. Franzen, D. S. Goodsell (2006) Tactile teaching: Exploring protein structure/function using physical models, Biochem. Mol. Biol. Educ. 34, 247–254. [6] G. A. Bain, J. Yi, M. Beikmohamadi, T. M. Herman, M. A. Patrick (2006) Using physical models of biomolecular structures to teach concepts of biochemical structure and structure depiction in the introductory chemistry laboratory, J. Chem. Educ. 83, 1322–1324. [7] K. Jittivadhna, P. Ruenwongsa, B. Panijpan (2009) Making ordered DNA and protein structures from computer-printed transparency film cut-outs, Biochem. Mol. Biol. Educ. 37, 220–226. [8] G. R. Parslow (2002) Commentary: Molecular visualization tools are good teaching aids when used appropriately, Biochem. Mol. Biol. Educ. 30, 128–129. [9] S. Bottomley, D. Chandler, E. Morgan, E. Helmerhorst (2006) jAMVLE, a new integrated molecular visualization learning environment, Biochem. Mol. Biol. Educ. 34, 343–349.