In the Laboratory
Structural Studies of Phycobiliproteins from Spirulina: Combining Spectroscopy, Thermodynamics, and Molecular Modeling in an Undergraduate Biochemistry Experiment
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Ann T. S. Taylor* and Scott E. Feller Department of Chemistry, Wabash College, Crawfordsville, IN 47933; *
[email protected] In recognition of the integration of biochemical applications in the traditional areas of chemistry, the emerging opportunities for students in fields located at the interface of chemistry and biology, and the upcoming requirements from the ACS Committee on Professional Training, our department has recently reorganized its curriculum to incorporate a onesemester biochemistry course with laboratory as a requirement for all chemistry majors. To accommodate this additional course within the college’s limit on the total number of courses that can be required, the physical chemistry requirement was reduced from two semesters to one. To ensure that all students graduate with a solid understanding of physical chemistry principles, a plan was developed to incorporate and reinforce these principles throughout the curriculum, in courses both before and after the physical chemistry semester. In this article we describe a laboratory activity, crossing sub-disciplinary boundaries, that may be useful for other chemistry programs that are addressing these same issues. A common theme in biochemistry is the importance of three-dimensional structure in determining protein function. The advent of programs such as Kinemage, RasMol, Chime, and more recently, Protein Explorer, allows students to routinely visualize and manipulate protein structures. Furthermore, many biochemistry textbooks are packaged with a compact disc that includes protein structure files and viewing programs (1). In addition to these freely available protein viewing programs, many students have used commercial molecular modeling programs such as PC Spartan Pro and Hyperchem in general and organic chemistry. While a number of methods for integrating structure-viewing activities into the biochemistry classroom have been proposed (2, 3), molecular viewing and modeling have not been as extensively incorporated into the biochemistry or biophysics laboratory. The combined use of computer modeling and laboratory work allows students many of the same benefits as their use in the classroom, for example, the opportunity to connect measurable macroscopic properties with changes at the atomic level. In this laboratory, students correlate the structural changes that occur upon the denaturation of phycobiliproteins with changes in the absorption spectra and structure of the covalently bound cofactor, phycocyanobilin. Numerous laboratory experiments evaluating the denaturation of proteins including chymotrypsin (4), ribonuclease (5), and myoglobin (6, 7) have been described in the literature. Recently two qualitative experiments focusing on various conditions that cause denaturation of phycobiliproteins were published in this Journal (8, 9). While using the same proteins, this experiment differs in that it utilizes a single denaturant, includes a quantitative determination of the free energy of unfolding, and integrates molecular visualization and modeling components.
The availability, ease of purification, prior characterization of folding stability, and conformational change in the chromophore make phycobiliproteins ideal for an integrated protein-folding and modeling exercise. Blue-green algae such as Spirulina contain large amounts of phycobiliproteins. The phycobiliproteins are organized into a light-harvesting complex called the phycobilisome, which absorbs light, then transmits it to the photosystem II complex (10). Unlike most other antenna complexes, phycobiliproteins are highly water soluble, making them easy to use in the undergraduate laboratory. Phycobiliproteins have a chromophore, phycocyanobilin, covalently linked through a cysteine residue (Figure 1). The native protein structure constrains the chromophore to a planar conformation, maximizing the orbital extent (and thus its wavelength of maximum absorbance). Upon denaturation of the protein, the phycocyanobilin is no longer constrained to a planar conformation, and the absorbance at high wavelengths decreases substantially while the absorbance at near-UV wavelengths increases. This conformational change illustrates the importance of protein structure on cofactor conformation, and also demonstrates the effect of changing the extent of conjugation within the “particle in a box” model of conjugated systems. The combination of spectroscopy, thermodynamics, molecular visualization, and computational modeling allows students to simultaneously observe these phenomena on both a macroscopic and microscopic level. Experimental Details Students are provided a stock solution of approximately 10 mg phycobiliproteins per mL prepared from Spirulina (Sigma) as previously described (9), and an 8 M stock solution of urea in 0.1 M phosphate buffer at pH 7. Students select denaturant concentrations between 0 and 8 M, with a majority of the samples in the unfolding transition region of 2 to 6 M, while the protein concentration is held constant. Sample tubes with total volume of 2 mL are prepared with HOOC COOH
NH
H N
N
O
CH3 H 3C
H N O
CH S
Cys (protein)
Figure 1. Structure of phycocyanobilin.
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In the Laboratory
Hazards Urea is a skin irritant; affected skin should be rinsed copiously with water.
in Figure 2. In the absence of denaturant, a strong absorbance is observed at 625 nm with a shoulder at 650 nm, corresponding to slight variations in the planarity of phycocyanobilins in phycocyanin and allophycocyanin. As increasing amounts of urea are added, the absorption intensity at 625 nm decreases, while absorption at 350 nm (corresponding to phycocyanobilin associated with unfolded protein) increases. From the absorbance data, the equilibrium constant, Keq, for unfolding at each denaturant concentration is determined using a simple two-state model for the protein,
K eq =
Typical results for the absorption curve of the phycobiliprotein upon denaturation with urea are displayed 1468
=
Af − A A − Au
(1)
0.8 0.7
0.00 M 3.00 M
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Results and Discussion
[unfolded ] [folded ]
where Af is the absorbance at 625 nm of the completely folded protein, Au is the absorbance at 625 nm of the completely unfolded protein, and A is the absorbance at 625 nm measured at a specified denaturant concentration. The values of Af and Au are determined from the plateau regions at low and high denaturant concentrations, respectively. The Gibbs free energy upon unfolding can be obtained from the equilibrium constants, ∆Gc⬚ = ᎑RT lnKeq, where the subscript “c” emphasizes that the Gibbs free energy difference between protein conformations is a function of denaturant concentration. Assuming that the conformational Gibbs free energy difference is a linear function of denaturant concentration, c, in the transition region, ∆Gc⬚ = ∆GH2O⬚ − mc, allows the Gibbs free energy difference in the absence of urea to be determined from the y intercept of a graph of ∆Gc⬚ versus c. Typical values for ∆Gc⬚ in the transition range (i.e., where Keq is between 0.1 and 10) are graphed as a function of denaturant concentration in Figure 3. From this graph, the stability of the folded protein under nondenaturing conditions was determined by extrapolating to zero denaturant. For a more thorough discussion of the two-state model and spectroscopic approach employed in this analysis, the reader is referred to recent
Absorbance
0.1 mL stock protein solution, the appropriate volume of 8 M urea, and the balance of buffer solution. It is important that the protein is added last, after the buffer and urea stock solutions are well mixed. The samples are incubated for 15 minutes at room temperature (~22 ⬚C for the results presented here) before acquisition of absorbance spectra between 300 and 700 nm. The absorbance at 625 nm is recorded for subsequent analysis of the free energy difference between the folded and unfolded states. Students examine the interactions of the phycobiliprotein and phycocyanobilin from the structure of allophycocyanin available in the protein data bank (PDB; accession code 1ALL) using Protein Explorer. While phycocyanin makes up a greater proportion of phycobiliproteins in Spirulina, the structural alterations and interactions are more obvious in allophycocyanin (11). Protein Explorer is based on RasMol and Chime, and is freely available at http://www.proteinexplorer.org (accessed Sept 2002). Prior to this experiment, students completed an exercise that familiarized them with Protein Explorer’s basic commands. Significant advantages of Protein Explorer include pull-down menus, information about commands, and multiple levels of complexity, which make it easier for students to learn. Possible interactions between the protein and chromophore are mapped, and the dihedral angles of the phycocyanobilin backbone measured. The electronic structure of phycocyanobilin is then investigated using the program PC Spartan Pro (Wavefunction) by importing an edited version of the 1ALL PDB file. Students are provided this file where the atom records of the protein are removed, leaving only the phycocyanobilin structure that is bound to the ␣ subunit of the phycobiliprotein. The PDB structure, determined by X-ray crystallography, provides the positions of only the heavy atoms. PC Spartan Pro will automatically place the majority of the hydrogen atoms that are lacking in the PDB structure; however, students need to then inspect the structure to place the remaining hydrogens and ensure that the correct hybridization is assigned to each atom. Having thus obtained the structure of folded phycocyanobilin—the planar chromophore associated with the native structure of the protein—the energy of the to * transition is approximated by the difference in HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies obtained from a single point energy calculation at the semiempirical level using the AM1 model. To simulate the conformational changes upon denaturation of the protein, the structure is energy minimized via a molecular mechanics calculation employing the Merck Molecular Force Field. This unfolded conformation—the nonplanar chromophore in the unfolded, denatured protein— is then analyzed in a similar fashion to the folded state, the backbone dihedral angles measured, and the HOMO and LUMO energies calculated.
350
400
450
500
550
600
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λ / nm Figure 2. Denaturation curves for phycobiliprotein as the concentration of urea is varied.
Journal of Chemical Education • Vol. 79 No. 12 December 2002 • JChemEd.chem.wisc.edu
In the Laboratory
articles in this Journal (6, 7). The experimentally determined Gibbs free energy difference was highly reproducible in student hands (10.2 ± 2.8 kJ/mol, n = 6) and correlated well to literature values of 10–36 kJ/mol (12). The broad range of published values is due to the evaluation of phycobiliproteins from a wide variety of blue-green algae species with different ambient environmental temperatures (the fact that the student data are towards the low end of the published value range reflects that Spirulina grows at room temperature). During the molecular visualization exercise, students observe details of the protein structure. Allophycocyanin is a dimer of two alpha-helical proteins, with the phycocyanobilin fitting into a pocket on the surface of the protein, and the carboxylic acid groups interacting with the solvent. By using the “display contacts” command (a feature unique to Protein Explorer), students can view the amino acid residues of the proteins that potentially interact with the chromophore. Students can also measure the dihedral angles of the chromophore backbone and discover that except for one, the dihedral angles are close to 0⬚ or 180⬚, indicating a very nearly planar structure. The modeling with PC Spartan Pro offers students a second opportunity to visualize the phycocyanobilin structure, as it also allows free rotation of the molecule and the ability to measure geometric parameters such as dihedral angles. Additionally, PC Spartan Pro provides molecular orbital calculations that demonstrate clearly the character of the HOMO and of the LUMO. From the difference in the HOMO and LUMO energy eigenvalues, students estimate the wavelengths of maximum absorption for the bound and free phycocyanobilin in the native and denatured proteins, respectively. The computations confirm the experimental observation of increased energy-level spacing upon denaturation of the protein; however, the values of the calculated wavelengths do not correspond to transitions in the visible region of the electromagnetic spectrum. This shortcoming is typical of the simplistic approach taken here whereby the excitation energy is equated with the difference in ground state
orbital energies. Qualitative aspects of the effect of conformation on the spectroscopic results are obtained, that is, increased absorbance at lower wavelength and a corresponding decrease at higher wavelength; however, quantitative agreement would require more sophisticated methods that are both computationally expensive and beyond the scope of routine undergraduate studies. Comparing the conformations of the constrained and unconstrained phycocyanobilin in the native and denatured proteins, respectively, shows substantial changes in two dihedral angles, resulting from the dramatic movement of one of the pyrrole rings relative to the backbone. Thus the modeling studies provide, through consideration of both conformational energetics and electronic structure, an atomic-level picture of the observed wavelength shift. Numerous extensions to this exercise are possible. Other denaturants, such as guanidine hydrochloride, could be used, as could phycobiliprotein obtained from different cyanobacteria. The free energy of unfolding could also be followed through the increased absorbance at 325 nm or the decreased absorbance at 650 nm. If temperature control is available, the free energy difference between native and denatured states could be decomposed into enthalpic and entropic contributions. Another advantage of the phycocyanobilin system is that its fluorescence spectrum can be used to monitor the unfolding process, and actually provides a more sensitive measure of the relative concentrations of folded and unfolded states. In the computational section, more extensive conformational searching could be employed to model the free phycocyanobilin, or, as mentioned previously, more sophisticated electronic structure theory could be applied to this problem so that more than qualitative agreement between the wavelength shifts could be obtained. In conclusion, this activity integrates spectroscopic and molecular modeling methods to illustrate the importance of protein folding in the function of a photosynthetic pigment. Depending upon the emphasis of the instructions, this activity is appropriate for biochemistry, biophysics, and physical chemistry laboratory sequences. Acknowledgments This work was supported by Wabash College through the Haines Fund for the Study of Biochemistry, and the National Science Foundation through grants DUE-9952381 and MCB-0091508.
10.0
∆G o 兾 (kJ mol᎑1)
7.5
WSupplemental
Material
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PDB files for allophycocyanin and phycocyanobilin, as well as a student handout including background information and detailed instructions, are available in this issue of JCE Online.
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Literature Cited
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[urea] / (mol L᎑1) Figure 3. Gibbs free energy difference between native and denatured states as a function of urea concentration.
1. For example, Voet, D.; Voet, J. G.; Pratt, C. W. Fundamentals of Biochemistry and the accompanying CD, Biochemical Interactions; John Wiley & Sons: New York, 1998. 2. Weiner, S. W; Cerpovicz, P. F.; Dixon, D. W.; Harden, D. B.; Hobbs, D. S.; Gosnell D. L. J. Chem. Educ. 2000, 77, 401. 3. Cox, J. R. J. Chem. Educ. 2000, 77, 1424.
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9. Heller, B. A.; Gindt, Y. M. J. Chem. Educ. 2000, 77, 1458. 10. Glazer, A. N. J. Biol. Chem. 1989, 264, 1. 11. Brejc, K.; Ficner, R.; Huber, R.; Steinbacher, S. J. Mol. Biol. 1995, 249, 424. 12. Chen, C.-H.; Berns, D. S. Biophys. Chem. 1978, 8, 203; Chen, C.-H.; Kao, O. H. W.; Berns, D. S. Biophys. Chem. 1977, 7, 81.
Journal of Chemical Education • Vol. 79 No. 12 December 2002 • JChemEd.chem.wisc.edu
