Isothermal Titration Calorimetry and Macromolecular Visualization for

Laboratory Experiment pubs.acs.org/jchemeduc

Isothermal Titration Calorimetry and Macromolecular Visualization for the Interaction of Lysozyme and Its Inhibitors Chin-Chuan Wei,* Drake Jensen, Tiffany Boyle, Leah C. O’Brien, Cristina De Meo, Nahid Shabestary, and Douglas J. Eder† Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, Illinois 62026-1652, United States S Supporting Information *

ABSTRACT: To provide a research-like experience to upper-division undergraduate students in a biochemistry teaching laboratory, isothermal titration calorimetry (ITC) is employed to determine the binding constants of lysozyme and its inhibitors, N-acetyl glucosamine trimer (NAG3) and monomer (NAG). The extremely weak binding of lysozyme/NAG is determined using a competitive binding assay. Such interactions between lysozyme and its inhibitors are visualized with PyMol software, by which the hydrogen bond formation in the complexes is used to explain the binding specificity. The hydrogen bond inventory in the binding interface correlates with the heat enthalpy determined or derived from ITC measurements. A possible explanation for such a correlation is presented and used for an extensive discussion in thermodynamics and ligand−receptor interactions. This laboratory exercise stimulates students’ critical thinking about weak/strong binding interactions and the relationship between thermodynamics and structural changes. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Biochemistry, Biophysical Chemistry, Calorimetry/Thermochemistry, Molecular Modeling

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agent to kill invading Gram-positive bacteria. Lysozyme binds with the maximal six repeated N-acetylglucosamine oligosaccharides (NAG6). Lysozyme hydrolyzes NAG6 to NAG4 and NAG2 as the major products, and to two NAG3 molecules as the minor product. The resulting products remain bound to lysozyme and can inhibit enzymatic activity. ITC studies of the hen egg white (HEW) lysozyme/NAG3 complex have been documented.4,6 NAG and NAG2 also bind to lysozyme, albeit with much lower binding affinity. Measuring extremely low binding constants by a direct titration possesses difficulties, such as the large amounts of ligand and receptor molecules required to achieve an acceptable signal and reach binding saturation. However, this problem can be solved by using a competitive binding assay, in which a strong-binding ligand is titrated to the receptor in the presence of a weak-binding ligand. A decrease in apparent binding affinity (Kapp) is observed since the strongbinding ligand has to compete for the same binding site occupied by the weak-binding ligand.7 The binding of lysozyme and its inhibitors can be viewed using visualization software.4,8 PyMol produces high quality, three-dimensional images and can be integrated with other calculation tools to understand protein folding and ligand− receptor interactions. Here, PyMol is used with an experimental ITC exercise to enhance student learning for molecular recognition.

iomolecular recognition, including hormone−receptor, antibody−antigen, and protein−DNA recognition, is crucial for signal transduction, immune response, and gene regulation. Though such recognition is introduced extensively in biochemically related courses to explain biological responses, few students clearly understand how the strength of the interaction (binding affinity) is determined. One gold standard technique for determining binding constants is isothermal titration calorimetry (ITC). ITC does not require chemical modification and allows for direct measurements of heat change from the association of a ligand to its receptor, which is used to determine the association binding constant (Ka) and enthalpy difference (ΔH°). The Gibbs free energy difference at the standard state, ΔG°, becomes linked to Ka as ΔG° = −RT ln(Ka).1 The conventional entropy change (ΔS°) can be derived from the thermodynamic relationship ΔG° = ΔH° − TΔS°. ITC is becoming an important tool in modern biochemical research2,3 and undergraduate education.4,5 In teaching laboratories, ligand/receptor binding is chosen to be moderate so that the binding affinity can be determined by a direct titration. However, such an approach does not address the problems of measuring extremely strong and weak binding constants encountered in research projects. The binding specificity can typically be rationalized by hydrogen bond formation between ligand and receptor; however, it is challenging to relate thermodynamic parameters to possible structural changes. The lysozyme system was chosen because a wealth of research results are available for implementation and discussion. Animals, insects, and plants use lysozyme as a primary © XXXX American Chemical Society and Division of Chemical Education, Inc.

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DOI: 10.1021/ed5002569 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Figure 1. ITC results for lysozyme/NAG3 and lysozyme/NAG. The heat rate change was observed during titration of NAG3 to lysozyme (A) in the absence and (C) in the presence of NAG. Panels B and D show plots of heat evolved per injection versus molar ratio (i.e., [NAG3]/[lysozyme]) from panels A and C, respectively. The fitting results are given in Table 1. Since the fitting from NanoAnalyze does not provide parameter error estimations, the obtained data were further analyzed by MicroCal Analysis software (see Supporting Information) to provide the estimated statistical errors for individual experiments. The analysis yields n = 0.97 ± 0.01, Ka = 1.22 ± 0.09 × 105 M−1, and ΔH° = 47.03 ± 0.14 kJ/mol for panel C and n = 0.98 ± 0.01, Ka = 6.71 ± 0.05 × 104 M−1, and ΔH° = 35.79 ± 0.14 kJ/mol for panel D.

Table 1. ITC Binding Affinities and Thermodynamic Parameters for 2.4 mM NAG3 plus 0.14 mM Lysozyme Reactionsa Mean ± Standard Deviation Values Calculated Collectively from Student Data in 2010−2013, N = 31 Kapp, M−1

NAG Concentration, mM 0 5 10 20

1.31 1.16 1.01 6.75

± ± ± ±

0.11 0.10 0.14 0.10

× × × ×

ΔH° or ΔHapp, kJ/mol 5

10 105 105 104

−50.30 −42.10 −39.48 −35.60

± ± ± ±

2.21 2.51 2.35 2.10

ΔG°, kJ/molb

ΔS°, J/mol·Kb

−29.19 ± 0.23 −c −c −c

−70.80 ± 9.00 −c −c −c

n 0.96 0.93 0.96 1.07

± ± ± ±

0.14 0.06 0.16 0.18

The values were directly determined by ITC measurements at 25 °C and analyzed by NanoAnalyze. The values are presented as mean ± standard deviation calculated collectively from laboratories in 2010−2013. bThe values were calculated using these equations: ΔG° = −RT ln(Ka) and ΔS° = −(ΔG° − ΔH°)/T. cNot applicable for competitive binding experiments. a

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EXPERIMENTAL PROCEDURE The experiment consists of two sections: ITC and PyMol. The ITC project is conducted in teams of four, in which individual groups of 3 or 4 students perform their experiments using four different concentrations of NAG. Once the entire class finishes the experiment, their data are shared to allow for data analysis. Students spend ∼1 h in sample handling, and the instrument takes an additional 2.5 h to complete the titration. During the waiting period, students perform macromolecular visualization using the educational version of PyMol.9 The PDB IDs for lysozyme/NAG3 and lysozyme/NAG, 1HEW10 and 1JA7,11 respectively, can be accessed from the Protein Data Bank.12 The titration is performed using an ITC instrument by a series of 25 injections consisting of NAG3 (10 μL 2.4 mM) into lysozyme (0.14 mM) in 0.1 M, pH 5.0 phosphate or acetate buffer at 25 °C. Raw data are corrected for the heat of dilution for the titrant, which is predetermined by an instructor. NanoAnalyze software is used for fitting. Competitive experi-

ments are conducted under identical conditions, except that both lysozyme and NAG3 solutions contain NAG (5, 10, or 20 mM). The Ka for lysozyme/NAG (KNAG) is determined from eq 1.13 KNAG3 = 1 + KNAG[NAG] K app

(1)

where KNAG3 is the Ka for lysozyme/NAG3 and Kapp is the apparent binding constant determined in the presence of a specific concentration of NAG.

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HAZARDS Solid sodium hydroxide, acetic acid, and concentrated phosphoric acid used in buffer preparation are corrosive and can cause severe burns. Lysozyme may cause skin, eye, and digestion tract irritation. NAG and NAG3 are not hazardous according to OSHA criteria. The needle of the sample loading syringe is sharp B




and should be used with caution. All students must wear safety eye protection and gloves.

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Box 1. Modeling Exercise Questions (a) How many α helix and β sheet secondary structures exist in the lysozyme/NAG3 structure? (b) Based on Structural Classification of Proteins (SCOP),19 which category does lysozyme belong to? (c) How many cysteine and cystine residues are contained in lysozyme? (d) Where are the binding subsite(s) of lysozyme for NAG3 and NAG? (e) With a literature search, determine the catalytic amino acid residues and highlight them in the structure. (f) What amino acid residues of lysozyme form hydrogen bonds to NAG3 and NAG? Include the parts of amino acids and inhibitors which participate in the binding. (g) Are there any significant structural changes in lysozyme/ NAG3 and lysozyme/NAG?

RESULTS AND DISCUSSION

Isothermal Titration Calorimetry

The experiment has been completed by 31 students four times. The preparation and loading of samples were emphasized because those materials are limited and extra care is needed for proper handling of protein samples. Representative students’ data for the lysozyme/NAG3 experiment are illustrated in Figure 1. The data from the fitting were collected in a 3−4 year period and analyzed to provide a mean value and standard deviation. The values for ΔH° and Ka were determined to be −50.30 ± 2.21 kJ/mol and 1.31 ± 0.11 × 105 M−1, respectively, which agreed with published values.4,6 Titration of NAG3 into lysozyme in the presence of 20 mM NAG is shown in Figure 1C. The heat generated from individual injections decreased and additional NAG3 was required to saturate the binding. By changing the concentrations of NAG and determining their corresponding Kapp values (Table 1), KNAG was determined to be 53 ± 12 M−1 from three trials collectively in 2011−2013 (Figure 2), consistent with data obtained from direct ITC titrations14,15 and other techniques.16−18 A representative data set from 2011 with KNAG = 61 M−1 is also presented in Figure 2.

a presentation model, such as stick, cartoon, or color, was assigned, enabling students to visualize the interesting parts of the molecule clearly. Representative student work is shown in Figure 3. The outline of the secondary structures of the lysozyme/NAG3

Figure 2. Relationship between the apparent binding constants and concentrations of NAG. Using the mean values of Kapp (open circle) from the individual NAG concentrations obtained in 2010−2013, the slope (solid line) for eq 1 (i.e., KNAG) was determined to be 47 M−1 (error = 7 and r2 = 0.98). To better show the uncertainty from students’ data, a standard deviation for each KNAG3/Kapp at different NAG concentrations is depicted with the error bars, indicating the range of the slope obtained from students. Note that the KNAG3 value for each trial significantly influences the determination of KNAG3/Kapp, and thus KNAG. For example, a representative data set (cross symbol) from 2011 was analyzed by a linear regression analysis (dashed line), yielding intercept = 0.92 (error = 0.07) and slope = 61 (error = 6 and r2 = 0.98), indicating an upper bound of KNAG. When three KNAG values separately determined from three trials in 2011−2013 were analyzed collectively, the analysis gave KNAG = 53 ± 12 M−1 (mean ± standard deviation).

Figure 3. Structures of lysozyme/NAG3 and lysozyme/NAG. (A) The complex with emphasis on the secondary structure: the disulfide bridges as formed by cysteine residues (yellow), NAG3, and two catalytic residues (Glu35 and Asp52). (B) The inhibitor NAG3 bound in the cleft of lysozyme. (C) The hydrogen bond network forming between NAG3 and the surrounding amino acid residues. (D) The alignment of lysozyme (green) to NAG3 (orange) and lysozyme (cyan) to NAG (red).

complex stabilized by disulfide bridges is shown in Figure 3A. NAG3 occupies A−B−C subsites and does not make contact with the catalytic residues (Glu35 and Asp52), as judged by the distance criteria of 3.4−4.2 Å for nonbonded atoms,20 making it a self-inhibitor. The specificity of the binding results from the extensive hydrogen bonds formed by the moieties of NAG3 to Asp59, Trp62, Trp63, Asp101, Asn103, and Ala107 (Figure 3C).10 Furthermore, two aromatic residues (Trp62 and Trp63) stack with the hydrophobic patch of NAG3, contributing to the

Macromolecular Visualization

Students were oriented to basic PyMol functions by following instructions from a handout (see Supporting Information) and then by exploring the software features, guided by specific questions (Box 1). In general, the specific parts of structures, such as the protein backbone, inhibitors, and residues, were grouped together to become the “selection”. For each selection, C




included a competitive binding experiment, providing a researchlike example, where the ITC results were correlated with the use of visualization software.

complex stability. When NAG3 is replaced by NAG, there is no significant conformational change in the protein backbone and ligand binding site21 as their root-mean-square distances (rmsd) are 1.26 Å for all atoms or 1.13 Å for backbone atoms, respectively (Figure 3D). NAG binds to the C or D subsite with three hydrogen bonds between NAG and protein residues: Gln57, Asp59, and Ala107. The binding specificity for lyszoyme/NAG3 is attributed to nonpolar interactions and the newly formed six hydrogen bonds (HB). The HB formation is believed to contribute predominately to the enthalpy changes.22 However, it is naive to make the above conclusion even when it is in accord with a variety of studies and model systems. To calculate thermodynamic parameters such as the enthalpy change, one has to calculate all interactions, including ionic, electrostatic, van der Waals, and polarization of the interacting groups, as well as solvation between the free and ligand-bound receptors. In fact, understanding the correlation between structural components and thermodynamics is currently beyond theoretical and experimental approaches. Despite this, a hydrogen bond inventory was used to rationalize the enthalpy changes for lysozyme/ NAG3 and lysozyme/NAG, for which the data correlates to the number of hydrogen bonds observed in the crystal structures. Our rationale for such a correlation is simply an alternative explanation,23 and the instructor should point out this fact in order to stimulate students’ critical thinking. Given no coupling to the protonation state of lysozyme in the complex at pH 5,23,24 the enthalpy change determined by ITC corresponds to the binding enthalpy difference, yielding an average value for a HB formed between lysozyme and NAG3 as ∼8 kJ/mol-bond, which lies within the range of the reported 8−13 kJ/mol for amide HB formation.25

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ASSOCIATED CONTENT

S Supporting Information *

Student Handouts for ITC and PyMol and Instructor Notes. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. † D.J.E.: Emeritus, Southern Illinois University Edwardsville, Edwardsville, IL 62026-2224.

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ACKNOWLEDGMENTS We thank Nicole Reynolds and Allison Tatro for their help with experimental designs. The ITC work is sponsored by NSF DUE-0941517, and macromolecular visualization work is supported by the University’s Excellence in Undergraduate Education award.

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REFERENCES

(1) Tellinghuisen, J. Achieving Chemical Equilibrium: The Role of Imposed Conditions in the Ammonia Formation Reaction. J. Chem. Educ. 2006, 83, 1090−1093. (2) Ababou, A.; Ladbury, J. E. Survey of the year 2005: literature on applications of isothermal titration calorimetry. J. Mol. Recognit. 2007, 20, 4−14. (3) Ladbury, J. E. Calorimetry as a tool for understanding biomolecular interactions and an aid to drug design. Biochem. Soc. Trans. 2010, 38, 888−893. (4) Hall, M. L.; Guth, C. A.; Kohler, S. J.; Wolfson, A. J. Advanced instrumentation projects for first-year biochemistry laboratory. Biochem. Mol. Biol. Educ. 2003, 31, 115−118. (5) Wadsö, L.; Li, Y.; Li, X. Isothermal Titration Calorimetry in the Student Laboratory. J. Chem. Educ. 2011, 88, 101−105. (6) Kumagai, I.; Sunada, F.; Takeda, S.; Miura, K. Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes. J. Biol. Chem. 1992, 267, 4608−4612. (7) Sigurskjold, B. W. Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal. Biochem. 2000, 277, 260−266. (8) Pembroke, J. T. Bio-molecular modelling utilising RasMol and PDB resources: a tutorial with HEW lysozyme. Biochem. Mol. Biol. Educ. 2000, 28, 297−300. (9) Molecular Graphics System, V., Schrödinger, LLC. http://pymol. org/educational/ (accessed Feb 2015). (10) Cheetham, J. C.; Artymiuk, P. J.; Phillips, D. C. Refinement of an enzyme complex with inhibitor bound at partial occupancy. Hen egg-white lysozyme and tri-N-acetylchitotriose at 1.75 Å resolution. J. Mol. Biol. 1992, 224, 613−628. (11) Von Dreele, R. B. Binding of N-acetylglucosamine to chicken egg lysozyme: a powder diffraction study. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2001, 57, 1836−1842. (12) RCSB Protein Data Bank Web. http://www.rcsb.org/pdb/ home/home.do (accessed Feb 2015). (13) Tse, J. K.; Giannetti, A. M.; Bradshaw, J. M. Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: Subpicomolar Kd determined using competition titration calorimetry. Biochemistry 2007, 46, 4017−4027.

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ASSESSMENT For effective learning, 2 weeks before the experiments, an online Background Knowledge Probe26 in Blackboard was used to survey students’ knowledge so the instructor could focus on subjects not understood by the majority of students. Those questions assessed their knowledge regarding ΔH°, ΔS°, calorimetry, and macromolecular structure. Pre- and Post-Readiness Assessment Tests (RATs)26 were used before and after the experiments, respectively. The Pre-RAT focused on ITC specifically and was used to evaluate student preparation. The Post-RAT was designed to assess students’ logical and critical thinking. Students were divided into groups in a separate class once the final data analysis was complete. In each group, a leading student prompted the discussion so that the knowledge was transmitted among them. The assessment did not focus on whether students gave correct answers; instead, it sought to determine whether reasonable statements could be generated when presented with various discussion topics. A total of 31 students from 2010 to 2013 were assessed, and the results and discussion can be found in the Supporting Information.

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CONCLUSION In biochemistry lecture, molecular recognition is typically explained using three-dimensional structures to show the weak interactions between ligand and receptor. However, there are no laboratory exercises that aimed to measure thermodynamic parameters that can be somewhat rationalized by structural change. Here, the integration of ITC with PyMol was used to aid student understanding in an efficacious manner. This experiment D




(14) Bjurulf, C.; Wadso, I. Thermochemistry of lysozyme-inhibitor binding. Eur. J. Biochem. 1972, 31, 95−102. (15) Cooper, A.; Johnson, C. M. Isothermal titration microcalorimetry. Methods Mol. Biol. 1994, 22, 137−150. (16) Studebaker, J. F.; Sykes, B. D.; Wien, R. A nuclear magnetic resonance study of lysozyme inhibition. Effects of dimerization and pH on saccharide binding. J. Am. Chem. Soc. 1971, 93, 4579−4585. (17) Gopal, S.; Ahluwalia, J. C. Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme. Biophys. Chem. 1995, 54, 119−125. (18) Ikeda, K.; Hamaguchi, K. The binding of N-acetylglucosamine to lysozyme. Studies on circular dichroism. J. Biochem. 1969, 66, 513− 520. (19) Hubbard, T. J.; Murzin, A. G.; Brenner, S. E.; Chothia, C. SCOP: a structural classification of proteins database. Nucleic Acids Res. 1997, 25, 236−239. (20) Li, A. J.; Nussinov, R. A set of van der Waals and coulombic radii of protein atoms for molecular and solvent-accessible surface calculation, packing evaluation, and docking. Proteins 1998, 32, 111− 127. (21) Blake, C. C.; Johnson, L. N.; Mair, G. A.; North, A. C.; Phillips, D. C.; Sarma, V. R. Crystallographic studies of the activity of hen eggwhite lysozyme. Proc. R. Soc. London, B 1967, 167, 378−388. (22) Rupley, J. A.; Butler, L.; Gerring, M.; Hartdegen, F. J.; Pecoraro, R. Studies on the enzymic activity of lysozyme, 3. The binding of saccharides. Proc. Natl. Acad. Sci. U.S.A. 1967, 57, 1088−1095. (23) Banerjee, S. K.; Rupley, J. A. Temperature and pH dependence of the binding of oligosaccharides to lysozyme. J. Biol. Chem. 1973, 248, 2117−2124. (24) Garcia-Hernandez, E.; Zubillaga, R. A.; Chavelas-Adame, E. A.; Vazquez-Contreras, E.; Rojo-Dominguez, A.; Costas, M. Structural energetics of protein-carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors. Protein Sci. 2003, 12, 135−142. (25) Habermann, S. M.; Murphy, K. P. Energetics of hydrogen bonding in proteins: a model compound study. Protein Sci. 1996, 5, 1229−1239. (26) Angelo, T. A.; Cross, K. P. Classrom Assessment Techiques: A Handout for College Teachers, 2nd ed.; Jossey-Bass: San Francisco, 1993.

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Isothermal Titration Calorimetry and Macromolecular Visualization for

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