Laboratory Exercise Myoglobin Structure and Function: A Multiweek Biochemistry Laboratory s Projectw Todd P. Silverstein* Sarah R. Kirk Scott C. Meyer Karen L. McFarlane Holman From the †Department of Chemistry, Willamette University, Salem, Oregon, 97301, From the Abstract We have developed a multiweek laboratory project in which students isolate myoglobin and characterize its structure, function, and redox state. The important laboratory techniques covered in this project include sizeexclusion chromatography, electrophoresis, spectrophotometric titration, and FTIR spectroscopy. Regarding protein structure, students work with computer modeling and visualization of myoglobin and its homologues, after which they spectroscopically characterize its thermal denaturation. Students also study protein function (ligand binding equilibrium) and are instructed on topics in data analysis (calibration curves, nonlinear vs. linear regression). This upper division biochemistry laboratory project is a challenging and rewarding one that not only exposes students to a wide variety of important biochemical laboratory techniques but also ties those techniques together to work with a single readily available and easily characC 2015 by The International terized protein, myoglobin. V Union of Biochemistry and Molecular Biology, 43(3):181– 188, 2015. Keywords: inquiry-based teaching; biophysical methods; protein structure function and folding; computational biology; active learning; laboratory exercises Introduction The relationship between protein structure and function has been a crucial aspect of biochemistry for the last 50 years or more. In the modern undergraduate biochemistry course, discussion of the isolation, characterization, structure, and function of proteins takes up over one-fourth of the typical one-semester course. Laboratory projects focusing on computer modeling of protein structure have been increasingly common in the literature since about 2000 [1–7]. Almost all biochemistry laboratory texts feature projects in which students isolate a protein from a natural source, purify it, and then assay its biochemical activity [8–15]. In addition to these independent projects that last 223 laboratory periods and are centered on individual w s Additional Supporting Information may be found in the online version of this article. Scott C. Meyer’s current address is: Department of Chemistry, Benedictine University, Lisle, IL, 60532 *Address for correspondence to: Department of Chemistry, Willamette University, Salem, OR 97301. E-mail: tsilvers@willamette.edu Received 25 July 2014; Accepted 2 November 2014 DOI 10.1002/bmb.20845 Published online 27 February 2015 in Wiley Online Library (wileyonlinelibrary.com) Biochemistry and Molecular Biology Education enzymes, several semester-long biochemistry laboratories devoted to a single protein or enzyme have been published [8, 16–25]. Although the thematic unity of working on one specific protein for the whole semester is attractive, not all important experimental methods can be applied to a single protein. Therefore, instead of designing an entire course around one protein, we decided to embed an extended protein structure/function project within our semester-long upper division laboratory course, Experimental Biochemistry I (CHEM 346). We devote six 3-h laboratory periods in this course to an extended project on myoglobin (Mb). The remainder of the course includes a number of other multilab period projects that introduce our students to experimental methods such as HPLC, GCMS, phospholipid membrane dynamics, laser light scattering, spectrophotometric and fluorimetric determination of enzyme activity, and electrochemical biosensors. The heme protein myoglobin is found in most muscle tissue. Like hemoglobin (Hb), Mb contains a heme-bound Fe(II) cation that can be oxidized to the Fe(III) form (metMb). For many decades it was believed that the main function of the Mb-heme-Fe(II) cofactor was to bind O2, as well as CO, N2, nitrite, and azide ligands. Recent evidence suggests that in fact, the major biological function of Mb may be to catalyze the nitrite/nitric oxide interconversion 181 Biochemistry and Molecular Biology Education in muscle [26, 27]. Unlike Hb, which is tetrameric and binds oxygen cooperatively with a Hill coefficient of n 5 2.8, Mb is monomeric and binds oxygen noncooperatively (n 5 1). Mb is easily extracted from muscle tissue and spectrophotometric characterization of the Fe-heme is facile, as it absorbs strongly in the visible and UV ranges. In fact, the visible absorption of heme-coordinated Fe(II) in Mb is responsible for the reddish appearance of fresh (reduced) meat, whereas the visible absorption of heme-coordinated Fe(III) in metMb is consistent with the brownish color of old (oxidized) meat [28, 29]. We describe here our six-period project, in which students isolate Mb from ground beef [29], spectrophotometrically characterize its oxidized and reduced forms [29], probe its ligand binding equilibrium [30], perform molecular modeling on its structure and that of its homologues [31], and use FTIR to characterize its secondary structure in the native and denatured forms [32]. We have based our extended Mb project on the four papers cited above [29–32], adapting and expanding the projects beyond what is described in the original source papers. Specifically, we have added new methods of analysis (e.g., SDS-PAGE), adapted to changes in molecular modeling websites, and added extensive quantitative data analysis and curve-fitting components. Student Learning Objectives: 1. Proficiency in carrying out the following biochemical techniques: centrifugation and protein isolation; column chromatography and SDS-PAGE (proteins); and use of protein structure databases 2. Strengthened understanding of the following biochemical topics: protein structure modeling, characterization, and denaturation; protein homology and evolution; and protein-ligand binding equilibrium 3. Increased competence in biochemical data analysis, including: x-y scatter plots and curve fitting (both linear and nonlinear regression); and calibration curves 4. Increased confidence and mastery in using the following instruments: UV-Vis and FTIR spectrophotometers; gel electrophoresis; and in silico computer modeling of protein structure 5. Enhanced ability to: employ critical thinking and scientific reasoning; speak and write effectively about scientific concepts. Teaching and Laboratory Procedures This extended laboratory project centered around Mb requires six 3-h laboratory periods, which include some inclass time for data analysis. Detailed instructions are provided in the Supporting Information. In laboratory Period 1, students extract Mb from ground beef, reduce, and oxidize portions of the sample, use a Sephadex column to “desalt” the excess oxidizing agent, and use UV-Vis absorbance to 182 characterize the reduced and oxidized samples. In Period 2 students employ SDS-PAGE to characterize the extracted samples. In Period 3, azide binding to metMb is examined via spectrophotometric titration. In Periods 4 and 5, students use online databases, websites, and software to characterize the structure of Mb and a selected novel homolog. In the final period, students employ FTIR spectroscopy to probe protein secondary structure in both the native and thermally denatured conformations. Students work in pairs throughout this experiment. Because use of the FTIR spectrophotometer takes up all 3 h of Period 6, we can only schedule use of the instrument if students are paired. During Periods 1 and 3, each student pair has access to its own small Thermo-Electron Genesys-10 UV-Vis spectrophotometer. In Period 2, all pairs mount their gels and run from a single electrophoresis power supply. In Periods 4 and 5, paired students sit adjacent to each other, but each works on his/her own desktop computer in our computer room. We have run this project six times, every spring since 2009. Course enrollment has varied from 7 to 20 students, averaging 10212. Period 1, Part IA: Myoglobin Extraction, Separation, and Spectral Characterization To begin the project, students extract Mb from ground beef in a single 3-h laboratory period, following a modification of the protocol of Bylkas and Andersson [29]. In this part of the project, students work from the original literature source; their laboratory manual contains screenshots of selected paragraphs taken directly from the Bylkas and Andersson paper (see Supporting Information). A portion of the extracted Mb is fully reduced from Fe(III) to Fe(II) with dithionite (turning deep red) and a separate portion is fully oxidized to Fe(III) with ferricyanide (turning brown). Following oxidation, the metMb is separated from excess ferricyanide using a Sephadex G-25 size-exclusion gel chromatography “desalting” column. The three forms of Mb (original extract, reduced, and oxidized) can then be characterized spectrophotometrically. Reduced Mb features O2 bound to the Fe(II) center in the heme. It has prominent peaks in the visible range at about 540 and 580 nm, and its Soret band is at 417 nm with e417 5 128,000 M21 cm21 [29]. The oxidized metMb has H2O bound to the Fe(III)heme; its visible peaks appear at 505 and 620 nm, and its Soret band is at 409 nm with e409 5 179,000 M21 cm21 [29]. Given the prominent changes in the wavelength and molar absorptivities of the Soret band absorbance peaks, it is straightforward to spectrophotometrically determine the concentrations of Mb and metMb, and monitor changes in Fe(II)/(III) redox chemistry and ligand binding. Period 2: SDS-PAGE In a subsequent 3-h laboratory period students use SDSPAGE to separate and visualize the protein components of both the original Mb extract, and the desalted metMb Myoglobin Structure and Function FIG 1 SDS-PAGE gel of Mb solutions (representative student results). Lanes 1 and 8: Novex protein standards; Lanes 2 and 9: PP1 protein standards; the three smallest standards are 10, 15, and 20 kDa; Lane 3: Mb (horse skeletal muscle, Sigma M-0630); Lane 4: supernatant of ground beef extraction; Lane 5: supernatant 1 dithionite; Lanes 6, 7, and 10: 10, 20, and 15 mL (respectively) of supernatant 1 ferricyanide, Sephadex G25-desalted. In Lanes 327 and 10, MW(Mb) 5 16.2 6 1.8 kDa; in Lanes 427 and 10, the Mb band accounts for 20 6 3% of the protein in each lane. column eluent. Here students are introduced to electrophoresis, including loading and running the gels, quantifying TM the bands (using GelDoc ), and calculating molecular weights and protein composition of each mixture (Fig. 1). Students determine the molecular weight of Mb in their mixtures and compare it to the literature value (17.6 kDa for Sigma M-0630); they also determine the percentage of Mb in each mixture (20 6 3%), showing that the desalting Sephadex G-25 column merely serves to remove the excess ferricyanide, but does not appreciably separate any of the proteins in the mixtures. Period 3, Part IB: Myoglobin Function: Ligand Binding Students perform a spectrophotometric titration to monitor the binding of azide to Fe(III) in metMb and characterize the azide/metMb binding equilibrium. This modification of the experiment described by Marcoline and Elgren [30] also takes a single 3-h laboratory period. By following changes in the 540 (or 580) nm peak, students can calculate r, the ratio of bound ligand to total protein, and [L]free, the equilibrium concentration of free (unbound) azide ligand. Fitting the r vs. [L]free plot to the equation for hyperbolic saturation [Eq. (1)] allows a best fit determination of the protein-ligand dissociation equilibrium constant, Kd, and the number of bound ligand molecules per protein, n. r5 n 11Kd =½Lfree 5 n½Lfree ½Lfree 1Kd (1) Typically, a linearized form of the results, such as a Scatchard plot or a double-reciprocal plot, would be used to determine n and Kd. However, it has been pointed out Silverstein et al FIG 2 MetMb/azide spectrophotometric titration (representative student results). Total initial [Mb] 5 16.1 mM; changes in metMb absorbance at 543 nm were followed upon addition of azide aliquots amounting to final [N3-] 5 2 – 948 mM. (a) r vs. [N3-]free fit to the equation for hyperbolic saturation gives Kd 5 35.6 6 2.0 mM, n 5 0.973 6 0.013, and R2 5 0.997. (b) The double-reciprocal plot, 1/r vs. 1/ [N32]free gives intercept 5 1.9 6 0.8, slope 5 21 6 3 mM21, and R2 5 0.83 for all 12 points (solid line); this yields calculated values of n 5 0.52 6 0.22 and Kd 5 11 6 5 mM. Omitting the two lowest concentration points (1.1 and 3.9 mM free azide) gives intercept 5 1.075 6 0.023, slope 5 32.4 6 0.5 mM21, and R2 5 0.998 (dashed line); this gives calculated values of n 5 0.930 6 0.020 and Kd 5 30.1 6 0.8 mM. A Scatchard plot of these data (r/[N32]free vs. r) gives similar large error in the two lowest concentration points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] many times that linearizing data often magnifies error in the derived parameters [33–35]. Students are therefore instructed to analyze their results using both linear regression of linearized data and nonlinear regression of the hyperbolic curve, and discuss which is more reliable. Students find that n does indeed equal one for azide binding to metMb, and Kd is in the 10–100 mM range (see Fig. 2), in agreement with the literature (4–80 mM) [36]. Figure 2b clearly shows how error is magnified in the doublereciprocal plot for the two lowest concentration points. Even omitting these points gives errors, relative to the 183 Biochemistry and Molecular Biology Education FIG 3 Mb structure comparison. (A) solved structure of sperm whale Mb, pdb file 1VXA; (B) Swiss model predicted structure of Mb from unicorn icefish (Channichthys rhinoceratus), UniProt file E5G628. Color key for amino acid side chain types at the protein surface: white 5 hydrophobic; red 5 negative; blue5 positive; pink 5 polar neutral; yellow 5 aromatic; green 5 proline. Note the heme binding cavity in the center of both structures; most of the surface directly above this cavity is identical in both (A) and (B). Besides these and other similarities, six key differences between the two forms of Mb are marked above. values derived from nonlinear regression, of 215% and 24.5% in Kd and n, respectively; the Scatchard plot is a little better, with errors of 29% and 21.7%, respectively. pare the 3D structure of original protein to that of its novel homolog. Periods 4 and 5 Part II: Myoglobin Three-Dimensional Structure: Native vs. Denatured Myoglobin Structure on the Web In 1998, Leon et al. published a biochemistry laboratory project in which students used internet websites and freeware to model protein structure [31].1 Leon et al. describe a project in which students select a protein whose structure has already been solved and deposited in the Protein Data Bank (PDB). They then carry out a series of four operations on the internet: (a) use search programs to find a “novel homolog,” i.e., a homologous protein whose structure has NOT yet been solved; (b) submit the amino acid sequence of the novel homolog to secondary structure prediction programs and compare secondary structures of the original protein and its novel homolog; (c) submit the amino acid sequence of the novel homolog to Swiss Model to obtain a predicted three-dimensional structure; and finally (d) com- 1 In order to increase the diversity of protein structures examined, we have half of the students start with myoglobin, a predominantly alpha-helical protein, and the other half start with the predominantly beta-sheet protein, lysozyme. 184 It takes students about 4.5 h to finish operations (a–d) listed above. We have our students use the final 1.5 h in laboratory period 5 to prepare figures for an oral presentation in which they carefully characterize the major differences and similarities between the original protein and its novel homolog. All of the websites described by Leon et al. have changed dramatically in the nearly 2 decades since the original publication. Some things have gotten easier. For example, in 1998 one had to visit three separate websites to obtain a protein’s structure coordinates (PDB), structure, and function information (UniProt/SwissProt), and perform homology searching (Blitz). Currently all of this information can be obtained from the PDB website and links therein. On the other hand, other aspects of the project have gotten more difficult. The protein visualization program recommended by Leon et al., Rasmac, is woefully outdated; in addition, a useful protein structure comparison program that we discovered around 2000, Protein Explorer, has not been supported since about 2006. However, an even better freeware program, PyMOL, has since come into wide use [2, 37238]. This program not only allows homologous proteins to be aligned and visualized, but it also has many different viewing modes that can be implemented. We encourage students to explore the capabilities of PyMOL on their Myoglobin Structure and Function own, but we require that they use at least three different views to compare their two proteins: van der Waals surface; electrostatic surface; and polarity/hydrophobicity (Fig. 3). The former two views are built into PyMOL, and the latter is provided by the “color_by_restype” script written by researchers at Queens University in Kingston, Ontario [39]. Structure of Native and Unfolded Myoglobin Characterized by FTIR In the sixth and final 3-h laboratory period of our Mb structure and function project, students use FTIR to observe spectroscopic differences in native, folded proteins that are predominantly alpha-helical (e.g., Mb) and those that are predominantly beta-sheet (e.g., lysozyme). Students can determine the actual percent helix vs. sheet by accessing structural information at the Protein Data Bank (pdb) website, and compare this to the FTIR spectroscopic results that they get for each protein. They then incubate these proteins at elevated temperatures and use changes in FTIR peaks to characterize the thermal denaturation process (Fig. 4a). This final sub-project is based on the experiment described by Olchowitz et al. [32]. We have made two major modifications in order to dramatically improve data analysis. First, we found that in order to get the expected sigmoidal changes in FTIR peaks with changes in temperature, careful baseline subtraction, and normalization is required. In Fig. 4a the spectra are baselined at 1700 cm21 and normalized at the a-helix amide I peak, 1651 cm21. We describe these processes in detail in our Supporting Information. Second, we encourage our students to analyze their results in order to obtain a denaturation temperature, Tden (also called TU or Tmelt in the literature); this is the temperature at which the equilibrium between native/folded protein and denatured/ unfolded protein features a 50/50 mixture. The denaturation equilibrium, in which the folded, native protein (N) denatures, and unfolds (U) can be characterized by an equilibrium constant (Kden 5 KU 5 [U]eq/ [N]eq, and by an enthalpy and entropy for the process, DH U and DS U. Given a change in some FTIR spectroscopic parameter y as the protein unfolds, the dependence of the denaturation equilibrium on T, TU, and DH U, is shown in Eq. (2) (see Supporting Information, Appendix 4): y5 yloT 1yhiT e 11e DH 1 U 1 R ðTU 2T Þ DH 1 U 1 R ðT U 2 T Þ Compound Danger Hazard rating NPFA hazard 5 Eye damage; skin 2, GHS eye irritation Harmful if swallowed, inhaled, damage 5 1 or absorbed through skin Sodium azide Fatal if swallowed, or GHS dermal absorbed through skin toxicity 5 1, GHS oral toxicity 5 2, GHS aquatic toxicity 5 1 Potassium Harmful to aquatic life GHS aquatic ferricyanide toxicity 5 3 Sodium dithionite (bisulfite) Common Pitfalls Project Section Part IB: Part IIA Part IIB Problem At lowest azide concentrations, calculated concentration of free azide is negative. Amino acid sequence differences between UniProt vs. PDB sites. Swiss Model fails Protein aggregates at high T Determining the effect of T on protein structure: (2) Using nonlinear regression to fit the data in Fig. 4 gives best fit values for both TU and DH U. Students can then compare their results to literature values for Mb unfolding in aqueous buffer: TU 5 81–83 C, DH U 5 100–130 kcal/mol [40242]. Student results suggest (Fig. 4b) that in D2O, Mb unfolds at a lower temperature (65–70 C) than it does in aqueous buffer, but with a DH U that is the same or higher (100–300 kcal/mol). Silverstein et al Hazards Solution Set these free azide concentrations to the minimum value, zero. Find and omit the N-terminal signal sequence. Select a different template For T 85 C, incubate for only a few minutes. Have students consider as many spectral changes as seems productive. We plotted the relative absorbances of three different peaks in Fig. 4b, but the shift in kmax of the amide I peak can also be informative [32]. Student Reports For Part I (Mb extraction/characterization and azide binding), students write informal lab reports in which they address questions focused on data analysis to hone their critical thinking skills. They use Beer’s Law to calculate Mb concentrations, and discuss how heme-iron oxidation alters 185 Biochemistry and Molecular Biology Education FIG 4 FTIR characterization of Mb thermal denaturation (representative student results). (a) Relative absorbance (Ak/A1651) of 60 mg/mL Mb in D2O, at eleven different temperatures, 22–87 C. Note the appearance, above 64 C, of shoulders at 1618 and 1680 cm21, and an aggregated protein peak at 1570 cm21. (b) The three wavelengths in the spectra in (A) that change dramatically with T are plotted: R shoulder, A1618/A1651 (black circles); L shoulder, A1680/A1651 (blue diamonds); and aggregated protein peak, A1570/A1651 (blue diamonds). Fitting the R shoulder points using Eq. (2) gives DH U 5 290 6 70 kcal/mol and TU 5 65.79 6 0.15 C; fitting the L shoulder and aggregated protein points gives DH U 5 120 6 50 kcal/mol and TU 5 68.3 6 1.2 C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] UV-Vis absorbance. Students use SDS-PAGE standard protein mobilities to prepare a calibration curve to determine molecular weights; they compare their Mb molecular weight to the literature value, and also establish the fraction of Mb in their samples. For Part IB, students use both linear and nonlinear regression to determine Kd and n for azide binding to metMb. For Part II (protein structure modeling and thermal denaturation) students prepare an oral presentation in which they carefully characterize the major differences and similarities between Mb and their chosen novel homolog. They use FTIR amide I peaks to distinguish between proteins that are primarily a-helix versus b-sheet, and to follow temperature-driven changes in secondary structure. Finally, students use nonlinear regression of their FTIR peak versus T data to determine DH U and TU and compare to literature values. 186 Student Feedback/Assessment We assessed this project (and the entire Experimental Biochemistry course sequence) in two ways: with a pre/post laboratory skills exam, and with a survey of student alumni. Of the 33 questions on the exam, 8 covered concepts related specifically to this protein structure laboratory project (e.g., protein separation, structure characterization, ligand binding). On these eight questions, student performance improved from 53% correct before the start of the course, to 85% correct at the end (and 91% after the second semester of the course sequence). In addition to these exam results, as instructors of the course, we observed consistently an increase in both skills and confidence level as students moved through the course. We carried out a survey of our alumni to assess the impact of this laboratory sequence on their perceived knowledge of techniques, biochemical topics, and instrumentation. Myoglobin Structure and Function We had 18 people respond out of a total 23 accessible Biochemistry Track alumni over 4 years. From the survey results, it was clear that this laboratory was successful in meeting our intended goals. Students felt confident in their understanding of all the major biochemical topics covered, as well as in their ability to analyze biochemical data. One student wrote the following about what s/he got out of the course: “I have a solid foundation for lab technique and good lab practices upon which to build, and have an ability to learn new protocols quickly to become both proficient and efficient with them.” Interestingly, while students rated highly their knowledge of all of the instruments that they employed in the course, they expressed the greatest confidence in their ability to use the instruments that they were repeatedly exposed to. In particular they rated the course highly for helping them to develop critical thinking skills and scientific reasoning (4.6 on a Likert scale of 1 to 5) and to speak about scientific concepts (4.5). Students rated satisfaction with their ability to write scientifically even more highly (4.8). In short, the results of our pre/post laboratory skills exam and our alumni survey allow us to conclude that this project did indeed achieve the learning objectives that we aimed for (see list above). Further Extensions We decided to focus our protein structure/function project on myoglobin because it is readily obtained (from ground beef and in purified form), easily characterized (by spectrophotometry), and has been used in a number of published biochemistry laboratory projects. There are undoubtedly many other proteins that fit this bill and could be substituted for Mb. We have on occasion included another interesting laboratory project dealing with protein denaturation. As published [43], this project uses various concentrations of guanidine hydrochloride to denature chymotrypsin. Fluorescence spectroscopy is then used to probe both the structure of the protein as well as its enzymatic activity; the unique aspect of this particular laboratory project is that students simultaneously assay changes in both protein structure and function as a result of denaturation. Although we have not adapted this technique to Mb, we believe that it would not be difficult. In fact, due to the intrinsic absorbance of the Fe(II/III) center in Mb, UV-Vis spectrophotometry could probably be used in place of fluorimetry. In order to probe the effect of denaturation on protein structure, one would simply follow changes in the UV-Vis (or fluorescence) spectrum with guanidine concentration. To probe the effect on protein function, one would select a few representative guanidine concentrations and perform azide binding titrations at each one. Presumably, as denaturation increases, the Kd for azide binding should also increase (weaker binding). Summary We have developed an extended project for an upper division biochemistry laboratory course that uses Mb as a cen- Silverstein et al tral theme to investigate several important experimental techniques, as well as the relationship between the structure and function of a protein. Over six 3-h laboratory periods, the students isolate Mb from muscle tissue, spectroscopically characterize the oxidation state of its heme moiety and its ligand binding, analyze the protein’s structure via molecular modeling programs, and track its thermal denaturation with FTIR. We have expanded and combined previously described experiments [29232], and also updated procedures to reflect changes in the availability of structure analysis software like PyMOL [2, 37, 38]. This project has the advantage of fulfilling two sometimes disparate goals in laboratory curriculum development: It introduces students to a wide variety of biochemical techniques, while at the same time organizing the project around a central, biologically relevant theme. Acknowledgements The authors thank Willamette University for its extensive long-term support of laboratory curriculum development, and our colleague Dr. Alison Fisher for her help with this manuscript. 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