Activity pubs.acs.org/jchemeduc
Using Myoglobin Denaturation To Help Biochemistry Students Understand Protein Structure Yilan Miao and Courtney L. Thomas*,† Department of Chemistry, Susquehanna University, Selinsgrove, Pennsylvania 17870, United States S Supporting Information *
ABSTRACT: Analyzing and understanding data directly from primary literature can be a daunting task for undergraduates. However, if information is put into context, students will be more successful when developing data analysis skills. A classroom activity is presented using protein denaturation to help undergraduate biochemistry students examine myoglobin structure. Student learning goals are to understand relationships involving molar absorption coefficients and light absorbance, quantum yield and fluorescence, guanidine hydrochloride and protein folding, as well as protein folding and spectral properties of aromatic amino acids. This activity was performed by 56 students over three years. Results show statistically significant gains in student learning and student reported increased understanding. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Collaborative/Cooperative Learning, Fluorescence Spectroscopy, Proteins/Peptides
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INTRODUCTION
PLAN AND LEARNING GOALS This activity was performed in a biochemistry course taught in the fall semester, which covers information from amino acids through protein structure to enzyme catalysis. As a prerequisite, students have completed two semesters of both general chemistry and organic chemistry. Students were placed in groups of three or four to facilitate discussion. An individual pretest, the group activity, and an individual post-test were completed within a 60 min class period. Many biochemistry texts use hemoglobin and myoglobin as detailed examples of how protein structure affects function.12,13 In the interest of simplicity, the single chain myoglobin was selected for this activity instead of the four-subunit hemoglobin. The learning goals for the activity are to help students understand myoglobin structure through (1) the relationship between molar absorption coefficient and light absorbance, (2) the relationship between quantum yield and fluorescence, (3) how guanidine hydrochloride affects protein stability, and (4) how protein folding affects amino acid spectral properties. While the activity presented here focuses on analysis of previously published data, students can collect some data themselves. Horse heart myoglobin (Sigma-Aldrich M1882) and guanidine hydrochloride (Sigma-Aldrich 8 M solution G7294) could be used to collect myoglobin absorbance spectra and myoglobin fluorescence spectra at various concentrations of guanidine hydrochloride.11 Alternatively, students could propose a hypothesis and design their own experiments. For example, students could compare myoglobin versus hemoglo-
Students pursuing science careers must learn to read and analyze primary scientific literature.1 This effort can be aided by using an activity where data are put into context. Journals have noticed this disconnect, and some have added introductions to research articles. For example, Science includes a “This Week in Science”, a set of brief introductions to select articles.2 Similarly, the Journal of the American Chemical Society (JACS) begins each issue with “Spotlights on Recent JACS Publications”.3 This section contextualizes the articles and key results. Another benefit of using an activity that supplements published data is to help students develop data analysis skills rather than blind acceptance of conclusions drawn by the author(s). Undergraduate students tend to believe scientific publications are infallible, and when asked to analyze a paper, they repeat the author’s conclusions. However, when presented with data from several publications, students must examine the evidence and draw their own conclusions. Previous efforts have been made to bring primary literature into the classroom.4−8 However, these publications use primary literature directly instead of developing a context-based activity that guides students through data analysis. One article presented an in-class activity, but only after students read the primary literature.9 A cohesive activity is presented here examining myoglobin structure and folding. Myoglobin crystal structure, absorption spectrum, and fluorescence spectra are presented in context to guide students in their data analysis.10,11 Students are challenged to use this information to develop a better understanding of myoglobin structure. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: January 13, 2017 Revised: July 31, 2017
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DOI: 10.1021/acs.jchemed.7b00035 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 1. Myoglobin (PDB 1MBO) and its heme group. (A) The protein is shown as a ribbon (New Cartoon in VMD) with the heme group colored by atom, the W7 side chain in green, and the W14 side chain in brown.10 (B) The Fe(II)-heme is shown with a bound O2.13 The pyrrole ring lettering scheme is shown.
bin denaturation, examine myoglobin renaturation, or examine denaturation/renaturation of other commercially available proteins, such as lysozyme. Thus, this activity could serve as a starting point for a much broader discussion and deeper understanding of protein structure.
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ACTIVITY The activity begins with background information on myoglobin (17 kDa, single chain, functions in oxygen transport).14,15 Since the fully folded protein is required for heme binding and oxygen transportation, myoglobin structure is directly linked to its function.14 This link between structure and function is evident in all proteins which allows the concepts from this activity to be applied more broadly. Students are introduced to the three-dimensional structure of myoglobin and the embedded heme group (Figure 1). Additionally, students use the Protein Data Bank (PDB) code for myoglobin (1MBO) to display and manipulate the protein using Visual Molecular Dynamics (VMD), allowing them to identify the domain structure of myoglobin (8-stranded α helix).16 Next, students are introduced to the aromatic amino acids in myoglobin and shown a table of absorbance values and molar absorption coefficients (Table 1). From Table 1, students note
Figure 2. Absorption spectrum of native metmyoglobin in 0.1 M phosphate buffer.11 Reprinted with permission from ref 11. Copyright 1997 American Chemical Society.
correct fluorophore. The ∼280 nm peak is from tryptophan light absorption (Table 1) while the ∼400 nm peak is assigned to the heme group.11 Since the absorbance data for heme are not provided, students must deduce this information. Fluorescence is briefly reviewed in the activity (Supporting Information), and students are given the equation for quantum yield (photons emitted/photons absorbed). On the basis of the data in Tables 1 and 2, students describe the trend regarding Table 2. Fluorescence of Aromatic Amino Acids in Neutral Aqueous Solutionsa
Table 1. Absorbance of Light by Aromatic Amino Acidsa Amino Acid Phenylalanine Tryptophan Tyrosine a
λmax (nm) 258 280 275
ε (M−1 cm−1)
Amino Acid
Emissionb λ (nm)
Quantum Yield (Φ)
195 5800 1475
Phenylalanine Tryptophan Tyrosine
282 348 303
0.04 0.20 0.14
a
See refs 17 and 18.
See refs 19 and 20. bExcitation at 254 nm.
absorption and emission wavelengths for the aromatic amino acids. A shorter, higher-energy wavelength of light is absorbed, and a longer, lower-energy wavelength is emitted since some energy is lost in the transfer process. Then students identify tryptophan as the amino acid that should exhibit the greatest fluorescence based on both a high molar absorption coefficient and high quantum yield. To further their understanding, students are asked “If an aromatic amino acid is excited by UV light and passes that energy onto a different molecule, will fluorescence be observed
that tryptophan absorbs the greatest amount of light based on it having the largest molar absorption coefficient. When comparing the aromatic amino acids, students recognize that absorbance peaks for phenylalanine and tyrosine will be lower, possibly even unobservable, when compared to that of tryptophan. Also, since tryptophan and tyrosine absorb light at similar wavelengths, their peaks overlap and tryptophan absorbance can mask the tyrosine peak. The myoglobin absorption spectrum is presented in Figure 2, and students are asked to assign each peak in the figure to the B
DOI: 10.1021/acs.jchemed.7b00035 J. Chem. Educ. XXXX, XXX, XXX−XXX
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fluorescence. A minor portion of the spectral change can be attributed to increased polarity around the tryptophan residue as the protein unfolds.23 However, the most significant spectral shift is primarily attributed to movement of tryptophan away from heme as myoglobin denatures. The highest concentration of GuHCl used in Figure 3 was 6.1 M. Students consider if tryptophan fluorescence would increase if GuHCl concentration exceeded 6.1 M. Students are instructed to make a new graph plotting fluorescence intensity against GuHCl concentration as the independent variable (xaxis). This plot demonstrates that, by 6.1 M GuHCl, a plateau has been reached. Thus, increasing the concentration of GuHCl would not increase tryptophan fluorescence since the protein is already fully denatured.
from the original amino acid?” Group discussion should focus on different processes by which excitation energy can be released. Once options are listed, students explain that fluorescence will not be observed when excitation energy is dissipated by resonance energy transfer or direct electron transfer to a second fluorophore.21 Students are also asked which amino acid in myoglobin can be most readily detected spectroscopically transferring energy to the heme group. Assuming overlap between donor emission spectra and acceptor absorption spectra, since tryptophan has the highest quantum yield, students select that option. Guanidine hydrochloride (GuHCl) is introduced as a substance used to denature proteins.22 Students are presented with the structure and asked to hypothesize how denaturation occurs. Most students suggest that GuHCl competes with the protein’s native hydrogen bonds leading to protein unfolding. The professor can inform students that, while hydrogen bonding was originally thought to be responsible, recent research suggests a different method for how GuHCl denatures proteins. Students consider other options utilizing a threedimensional model of guanidine. Eventually, students suggest guanidine hydrochloride could denature proteins through stacking interactions with aromatic amino acid side chains.22 Since this is an area that is actively researched, the class can discuss how new research results change the current understanding of biochemical processes. A figure showing myoglobin fluorescence spectra in the presence of increasing concentrations of GuHCl is presented (Figure 3). Students identify tryptophan fluorescence as the source of the ∼340 nm peak (Table 2).
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STUDENT LEARNING OUTCOMES To determine the effect of this activity on student learning, all students were given a pretest and post-test (Supporting Information). While the questions on both tests were the same, the order of the question and answer options was rearranged. Questions were designed to test student progress on the learning goals. Pretest and post-test averages and standard deviations are shown for all three years of data collection in Table 3. A paired Table 3. Comparison of Pretest and Post-Test Scores
a
Year
Pretest Average ± SDa
Post-Test Average ± SD
Paired t Test p Value
Number of Students (n)
2014 2015 2016
3.88 ± 1.53 4.40 ± 1.54 3.95 ± 1.18
8.88 ± 1.11 9.05 ± 0.83 9.21 ± 0.79
2.3 × 10−09 3.2 × 10−10 1.9 × 10−13
17 20 19
SD = standard deviation.
t test (2 tailed, n listed on Table 3) was used to determine if the pretest scores were statistically different from the post-test scores. For all three years, post-test scores were significantly higher than pretest scores (Table 3). This indicates that the activity was successful in increasing student knowledge. Students were also asked to rank their perceived understanding of topics covered in the activity. A five-point Likertlike scale (1 = strongly disagree, 2 = disagree, 3 = ambivalent/ uncertain, 4 = agree, 5 = strongly agree) was used. The questions and assessment results are shown in Table S4 (Supporting Information). The first five questions addressed understanding of myoglobin structure and function, extinction coefficient relation to light absorption, quantum yield relation to fluorescence, how GuHCl affects proteins, and how protein folding affects its spectral properties. The values highlighted in Table S4 show a statistically significant difference between the pretest and post-test means. The first five questions were used to determine the students’ perceived level of understanding regarding activity topics. In 2014 and 2016, students reported a significant increase in understanding of all five topics. In 2015, students reported a significantly increased understanding of myoglobin structure and function as well as how GuHCl affects proteins. However, 2015 students did not report a statistically significant increased understanding of the other topics. Because 2014 and 2016 students reported significant increases in understanding and all three groups were exposed to the same information in class prior to the activity, perhaps 2015 students were less confident regarding their level of understanding.
Figure 3. Fluorescence spectra of native metmyoglobin ( lowintensity) and metmyoglobin in 1.1 M GuHCl (---), 1.3 M GuHCl (dotted), 1.5 M GuHCl (-·-), and 6.1 M GuHCl ( high-intensity) where λex = 285 nm and λem = 340 nm.11 Reprinted with permission from ref 11. Copyright 1997 American Chemical Society.
Students examine Figure 3 and suggest what causes the fluorescence intensity to increase as the concentration of GuHCl increases. This requires students to recall that tryptophan absorbs and emits light at certain wavelengths as well as recognize that energy can be transmitted from tryptophan to neighboring fluorophores (heme). Eventually, they deduce that, as myoglobin unfolds, tryptophan moves away from the heme group. As the distance increases, tryptophan reduces energy transfer to the heme and increases C
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(5) French, L. G. Teaching Organic Synthesis. An Advanced Organic Chemistry Course That Uses the Primary Literature. J. Chem. Educ. 1992, 69 (4), 287−289. (6) Gallagher, G. J.; Adams, D. L. Introduction to the Use of Primary Organic Chemistry Literature in an Honors Sophomore-Level Organic Chemistry Course. J. Chem. Educ. 2002, 79 (11), 1368−1371. (7) Hoskins, S. G.; Stevens, L. M.; Nehm, R. H. Selective Use of the Primary Literature Transforms the Classroom Into a Virtual Laboratory. Genetics 2007, 176, 1381−1389. (8) Ferrer-Vinent, I. J.; Bruehl, M.; Pan, D.; Jones, G. L. Introducing Scientific Literature to Honors General Chemistry Students: Teaching Information Literacy and the Nature of Research to First-Year Chemistry Students. J. Chem. Educ. 2015, 92 (4), 617−624. (9) Murray, T. A. Teaching Students to Read the Primary Literature Using POGIL Activities. Biochem. Mol. Biol. Educ. 2014, 42 (2), 165− 173. (10) Phillips, S. E. Structure and Refinement of Oxymyoglobin at 1.6 Å resolution. J. Mol. Biol. 1980, 142 (4), 531−554. (11) Jones, C. M. An Introduction to Research in Protein Folding for Undergraduates. J. Chem. Educ. 1997, 74 (11), 1306−1310. (12) Garrett, R. H.; Grisham, C. M. Biochemistry, 5th ed.; Brooks/ Cole, Cengage Learning: Belmont, CA, 2013; pp 497−509. (13) Voet, D.; Voet, J. G. Biochemistry, 4th ed.; John Wiley & Sons: Hoboken, NJ, 2011; pp 323−354; p 324, Figure 10-1. (14) Hendgen-Cotta, U. B.; Kelm, M.; Rassaf, T. Myoglobin Functions in the Heart. Free Radical Biol. Med. 2014, 73, 252−259. (15) Dautrevaux, M.; Boulanger, Y.; Han, K.; Biserte, G. Structure Covalente de la Myoglobine de Cheval. Eur. J. Biochem. 1969, 11 (2), 267−277. (16) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14 (1), 33−38. (17) Edelhoch, H. Spectroscopic Determination of Tryptophan and Tyrosine in Proteins. Biochemistry 1967, 6 (7), 1948−1954. (18) Fasman, G. D. Handbook of Biochemistry and Molecular Biology 3rd ed.; CRC Press: Cleveland, OH, 1976; pp 183−203. (19) Teale, F. W. J.; Weber, G. Ultraviolet Fluorescence of the Aromatic Amino Acids. Biochem. J. 1957, 65 (3), 476−482. (20) Ghisaidoobe, A. B. T.; Chung, S. J. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Forster Resonance Energy Transfer Techniques. Int. J. Mol. Sci. 2014, 15, 22518−22538. (21) Consani, C.; Aubock, G.; van Mourik, F.; Chergui, M. Ultrafast Tryptophan-to-Heme Electron Transfer in Myoglobins Revealed by UV 2D Spectroscopy. Science 2013, 339 (6127), 1586−1589. (22) Lim, W. K.; Rosgen, J.; Englander, S. W. Urea, But Not Guanidinium Destabilizes Proteins by Forming Hydrogen Bonds to the Peptide Groups. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (8), 2595−2600. (23) Marques, J. T.; de Almeida, R. F. M. Application of Ratiometric Measurements and Microplate Fluorimetry to Protein Denaturation: An Experiment for Analytical and Biochemistry Students. J. Chem. Educ. 2013, 90 (11), 1522−1527.
The next four questions shown in Table S4 were used as a control between the pretest and post-test scores. The authors did not expect students’ assessments of their ability to read primary literature, their enjoyment of working through activities in class, their enjoyment of working in groups in class, or their estimation of typical grades earned to change between the pretest and post-test. As shown in Table S4, no statistically significant difference was observed between pretest and posttest answers to these questions for all three years. These data validate the reliability of the pretest and post-test results. To determine if students enjoyed the activity, the post-test included the final question on Table S4. Student responses were overwhelmingly positive with an average between “agree” (4) and “strongly agree” (5) for all three years.
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CONCLUSIONS Overall, this activity accomplished the goal of helping biochemistry students understand myoglobin structure and denaturation. Statistically significant increases in student learning were observed as well as increased understanding reported by students. This activity demonstrates how data from the primary literature can be contextualized to yield a productive and enjoyable educational activity.
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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.7b00035. Student handout, student quiz, comparison of student reported understanding of activity concepts, and answer key (PDF, DOCX)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Courtney L. Thomas: 0000-0002-7264-541X Present Address † Department of Chemistry, Bucknell University, Lewisburg, Pennsylvania 17837, United States.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge Susquehanna University for supporting activity development and testing. We also thank T. Wade Johnson for editing the manuscript.
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REFERENCES
(1) Smith, G. R. Guided Literature Explorations: Introducing Students to the Primary Literature. J. Coll. Sci. Teach. 2001, 30, 465−469. (2) Grocholski, B.; Hodges, K.; Nusinovich, Y.; Stern, P.; Stajic, J.; Sugden, A.; Riddihough, G.; Lavine, M.; Mueller, K.; Wigginton, N.; Riddihough, G.; Yeston, J.; Smith, K.; Vignieri, S.; Sugden, A.; Kiberstis, P.; Colmone, A.; Wong, W. This Week in Science. Science 2017, 355 (6320), 35. (3) Herman, C.; Su, X. Spotlights on Recent JACS Publications. J. Am. Chem. Soc. 2016, 138 (51), 16567. (4) Fikes, L. E. Advanced Organic Chemistry: Learning from the Primary Literature. J. Chem. Educ. 1989, 66 (11), 920−921. D
DOI: 10.1021/acs.jchemed.7b00035 J. Chem. Educ. XXXX, XXX, XXX−XXX
