In the Laboratory
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A Simple Protein Purification and Folding Experiment for General Chemistry Laboratory Robert Bowen, Richard Hartung, and Yvonne M. Gindt* Department of Chemistry, University of Nebraska at Kearney, Kearney, NE 68849; *
[email protected] Traditionally, general chemistry has concentrated on introductory inorganic chemistry. While simple inorganic compounds are useful to illustrate most of the concepts discussed in general chemistry, many of our students, especially those who are enrolled in the class as a prerequisite to a career in the health professions, view the simple compounds as irrelevant and uninteresting. They are more interested in the chemistry of biological systems. To meet the goal of including more biochemistry in our laboratories, we searched for an experiment that would allow for the inclusion of simple protein chemistry. We needed an experiment in which the protein to be studied can be isolated quickly using the tools present in our general chemistry laboratory. We also searched for a protein with which relevant concepts could be easily illustrated. In this paper we outline a laboratory that meets these goals. We use phycobiliproteins to study intermolecular forces and their relationship to protein folding, a topic appropriate for the end of the first semester of general chemistry. Unlike other protein-folding laboratories (1), this one allows the students to discern the folding state of the protein by simple color changes. Background Cyanobacteria, formerly known as blue-green algae, contain an elaborate light-harvesting antenna complex in addition to chlorophyll molecules. The complex is made up of approximately 300 chromoproteins or phycobiliproteins (2, 3). The phycobiliproteins absorb sunlight from 500 to 650 nm, a region of the solar spectrum in which chlorophyll doesn’t absorb. The energy absorbed by the phycobiliproteins is eventually converted into chemical energy by the photosystem II reaction center. Phycobiliproteins have several desirable properties. They are water soluble and exceedingly stable at room temperature. They are brightly colored and highly fluorescent. They comprise roughly half the dry weight of the cell, so they are very plentiful in the cell. Many health food stores and drug stores now sell dried Spirulina cells in capsule form as a protein source. This cyanobacterium contains two phycobiliproteins: phycocyanin and allophycocyanin. Phycocyanin, the major component of the light-harvesting antenna, absorbs 620–630-nm light; allo-
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phycocyanin absorbs 650-nm light. Both phycobiliproteins contain an open-chain tetrapyrrole chromophore, phycocyanobilin, which is covalently attached to the protein via a single thioether bond to cysteine (see structure; from ref 4 ). CO2−
O
N H
H N
CO2−
N
H N
O
CH2 S
Cys(Protein)
Phycocyanobilin
The color of the chromophore is strongly dependent upon its environment (5). In the native protein, the chromophore is held in a linear conformation by hydrogen bonding to nearby side chains; the protein acts as a scaffold to hold the chromophore in the desired position. If the protein is unfolded, the scaffolding of nearby side chains is removed from chromophore, which then folds into a cyclic lockwasher conformation. The absorption spectrum of the chromophore in the cyclic lockwasher conformation is very different from the spectrum of the linear conformation. Instead of absorbing in the visible region, the chromophore now absorbs at around 370–380 nm. Therefore, the chromophore acts to report the integrity of the protein structure. When in its native form, the protein has a beautiful dark blue color. When it is unfolded, its blue color fades away. In addition to absorption spectroscopy, fluorescence can be used to assay the integrity of the protein structure. When the tetrapyrrole chromophore is held in a linear conformation by the protein scaffolding, it has a fluorescence quantum yield of approximately 60% with a fluorescence maximum of 650 nm in phycocyanin (2). When the chromophore is allowed to assume the cyclic lockwasher position, the fluorescence disappears. Therefore, when the protein is unfolded, the red fluorescence normally apparent even in room light will disappear.
Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
In the Laboratory
The Experiment Because of these properties, this protein will help to illustrate the basic forces involved in protein folding. This topic is appropriate for further discussion of intermolecular forces in general chemistry. The folding of water-soluble proteins depends strongly upon the presence of co-solvents in the aqueous solution. A simple way to describe the act of protein folding is to use the following idea. To dissolve a protein in an aqueous solution, a cavity must be formed in the solvent (6, 7 ). If the surface tension of the solvent is high, it will be energetically unfavorable to form the cavity; therefore, the protein will assume the smallest volume possible, which is generally the native folding of the structure. If the surface tension of the aqueous solution is low, it will be more energetically favorable to form the cavity in which to place the protein. The protein can assume a more unfolded, or less native, structure. The students can add different co-solvents to water to increase or decrease the surface tension of the solvent, and using the simple idea discussed above, predict whether the protein will unfold or remain in its native conformation. The surface tension depends strongly upon the strength of the intermolecular forces between the water molecules and the co-solvent. Other possible mechanisms of protein denaturation are also discussed, including competition between protein amino acids and co-solvents for hydrogen bonding, action of a detergent, mechanical shock, and temperature. Some simplifications have been made in the presentation of the material to the students, but we believe the material is still correct. The procedure outlined below is intended to fill one 3or 4-hour laboratory period, and it is designed for students working in pairs. In the experiment, the students grind dried Spirulina cells with an equal mass of silica, using a mortar and pestle. The silica and cell fragments are separated from the water-soluble protein by centrifugation in a table-top centrifuge and filtration. The protein can be stored for several weeks at this stage in the procedure, although we suggest that an antimicrobial agent (such as 5 mM sodium azide) be added to protect the protein from bacterial growth. At this point, the students will have a deep blue solution, which emits red fluorescence that is readily visible under room lights. The students will use small fractions of the resulting solution to test the effects of different reagents on the protein’s structure. The level of sophistication of the experiment can vary here. If available, an absorption spectrometer or fluorimeter can be used to assay the extent of denaturation, but the color changes due to protein denaturation are easily seen by the eye. We have found 6.0 M urea (out-competes the protein for hydrogen bonding to water) to be a very successful denaturant, whereas 0.5 M sodium phosphate (high-surface-tension cosolvent) adjusted to pH 7 is good for keeping the protein folded. The denaturation process is reversible if the protein isn’t allowed to stand too long in the denaturant. To refold
the protein, dilute out the denaturant with phosphate buffer at neutral pH. Equipment and Materials The equipment used in this laboratory is readily available in most general chemistry laboratories. The cells used as the source of the protein can be found in most supermarkets or health food stores. The simple procedure makes phycocyanin a desirable addition to the general chemistry laboratory. Hazards The students need proper training on the use of centrifuges, to prevent injury. Urea is a skin irritant; spills onto the skin should be rinsed with copious amounts of water. Student Response We surveyed 48 of our students regarding the laboratory, with the following results. On a scale of 1 (strongly disagree) to 5 (strongly agree), the average response to the statement “This laboratory was interesting” was 4.2. To the statement “I would recommend this lab continue to be used in the future” the average response was also 4.2. To the statement “The lab helped me to understand concepts from lecture” the average response was 3.8. The last statement is somewhat misleading because approximately one-fifth of the students hadn’t covered intermolecular forces in lecture when they completed the laboratory. The instructors were pleased with the results of the laboratory. Acknowledgment Y.M.G. gratefully acknowledges support for initial work on the laboratory from a National Science Foundation ILI grant (DUE-50262). W
Supplemental Material
Background information, instructions and handouts for students, and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Jones, C. M. J. Chem. Educ. 1997, 74, 1306. 2. Glazer, A. N. Biochim. Biophys. Acta 1984, 768, 29–51. 3. MacColl, R.; Guard-Friar, D. Phycobiliproteins; CRC: Boca Raton, FL, 1987. 4. Szalontai, B.; Gombos, Z.; Csizmadia, V.; Bagyinka, C.; Lutz, M. Biochemistry 1994, 33, 11823–11831. 5. MacColl, R.; Berns, D. Isr. J. Chem. 1981, 21, 296–300. 6. Melander, W.; Horvath, C. Arch. Biochem. Biophys. 1977, 183, 200–215. 7. Baldwin, R. L. Biophys. J. 1996, 71, 2056–2063.
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