In the Laboratory
An Integrated Biochemistry Laboratory, Including Molecular Modeling Adele J. Wolfson,* Mona L. Hall, and Thomas R. Branham Department of Chemistry, Wellesley College, Wellesley, MA 02181 The dilemma of designing an advanced undergraduate laboratory lies in the desire to teach and reinforce basic principles and techniques while at the same time exposing students to the excitement of research. We report here on a one-semester, project-based biochemistry laboratory that combines the best features of a cookbook approach (high success rate, achievement of defined goals) with those of an investigative, discovery-based approach (student involvement in the experimental design, excitement of real research). Individual modules may be selected and combined to meet the needs of different courses and different institutions. The central theme of this lab is protein purification and design. This laboratory accompanies the first semester of biochemistry (Structure and Function of Macromolecules, a course taken mainly by junior and senior chemistry and biological chemistry majors). The protein chosen as the object of study is the enzyme lysozyme, which is utilized in all projects. It is suitable for a student lab because it is easily and inexpensively obtained from egg white and is extremely stable, and its high isoelectric point (pI = 11) allows for efficient separation from other proteins by ion-exchange chromatography. Furthermore, a literature search conducted by the resourceful student reveals a wealth of information, since lysozyme has been the subject of numerous studies. It was the first enzyme whose structure was determined by crystallography (1). Hendrickson et al. (2) have previously described an intensive one-month laboratory course centered around lysozyme, although their emphasis is on protein stability rather than purification and engineering. Lysozyme continues to be the focus of much exciting new work on protein folding and dynamics, structure and activity (3–5). This lab course includes the following features: (i) reinforcement of basic techniques, such as preparation of buffers, simple enzyme kinetics, and absorption spectroscopy; (ii) experience with methods of protein purification; (iii) incorporation of appropriate controls into experiments; (iv) use of basic statistics in data analysis; (v) writing papers and grant proposals in accepted scientific style; (vi) peer review; (vii) oral presentation of results and proposals; and (viii) introduction to molecular modeling. Figure 1 illustrates the modular nature of the lab curriculum. Elements from each of the exercises can be separated and treated as stand-alone exercises, or combined into short or long projects. We have been able to offer the opportunity to use sophisticated molecular modeling in the final module through funding from an NSFILI grant. However, many of the benefits of the research proposal can be achieved with other computer programs, or even by literature survey alone. The skills and knowledge required for protein purification and design are developed in three units: (i) an introduction to critical assays needed to monitor degree of purification, including an evaluation of assay param*Corresponding author. Email:
[email protected] Phone: (617) 283-3106
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eters; (ii) partial purification by ion-exchange techniques; and (iii) preparation of a grant proposal on protein design by mutagenesis. Brief descriptions of each of these units follow, with experimental details of each project at the end of this paper. Assays for Lysozyme Activity and Protein Concentration (4 weeks) The assays mastered during the first unit are a necessary tool for determining the purity of the enzyme during the second unit on purification by ion exchange. These assays allow an introduction to the concept of specific activity (units of enzyme activity per milligram of total protein) as a measure of purity. In this first sequence, students learn a turbidimetric assay for lysozyme activity and a colorimetric one for protein concentration. Familiarity with the assays is reinforced by an independently designed project to modify a variable in one of these assays. The assay for lysozyme activity is that of Shugar (6), based on hydrolysis of a cell-wall suspension from the bacterium Micrococcus lysodeikticus, a substrate that is particularly sensitive to lysozyme. As the cell walls are broken down by the enzyme, the turbidity of the sample decreases. This decrease can be conveniently measured by following the decrease in absorbance at a wavelength of 450 nm, using a spectrophotometer or other device for measuring light scattering. The Bradford method (7), a standard assay, is used to determine protein concentration. Using the data from both lysozyme activity assays and protein concentration assays, students can calculate the specific activity for commercial lysozyme and an eggwhite solution. These calculations clearly demonstrate the increase in specific activity with increasing purity, since the purified (commercial) preparation has a specific activity approximately 20-fold higher than that of the crude egg-white solution. Enzyme and Protein Assays
Independent assay project
Library research
Grant proposal
Protein purification
Computer modeling
Peer review
Oral presentation
Figure 1. Design of project-based biochemistry laboratory. Modules (projects, or portions of projects) are indicated as boxes. Each of these can be treated independently, or used as part of a larger project. Solid lines indicate some suggested paths from one module to the next.
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Lysozyme Purification by Ion-Exchange Chromatography (5 weeks) As suggested by Strang (8), students can design a rational purification of lysozyme using ion-exchange chromatography when presented with information on the isoelectric point of the enzyme and the properties of ion- exchange resins. One week is spent discussing protein purification and the relative advantages and disadvantages of different resins. Each group has a choice of anion-exchange (DEAE) or cation-exchange (CM) resins. Because lysozyme is positively charged below a pH of 11, it will not be adsorbed to an anion-exchange resin, but will be adsorbed to the cation-exchange resin. Therefore, for the cation-exchange protocols, there are further options for methods of collecting and eluting the desired protein. A purification table, including information on yield, specific activity, and degree of purification, is constructed. As described by both Strang (8) and Hurst et al. (9), excellent purifications can be obtained by cationexchange chromatography, especially when elution is carried out with a salt gradient. Even a stepwise elution yields a highly purified fraction. Students’ results for degrees of purification using cation exchange range from 15- to 50-fold. Anion-exchange has been less successful, with purification factors less than 5, although the principle that the positively charged lysozyme will not be adsorbed to the resin is still noted. During the last lab for this project, the crude and purified fractions are analyzed by SDS–polyacrylamide gel electrophoresis. The results of electrophoresis correlate well with calculated purification results. It is very clear that for preparations with high degrees of purification, a single band corresponding to standard lysozyme is observed, whereas for the anion-exchange procedures, multiple contaminating bands are still present. Allowing the students to choose and plan their own purification scheme in consultation with the instructor not only gives them experience in experimental design, but also introduces some flexibility in the level of work expected from each student. Those students who have had much previous lab experience in other courses or who have done independent research can choose to carry out a complex purification scheme, giving them exposure to techniques they may not have used before, whereas students with less experience can be guided to a less demanding purification procedure. Grant Proposal on Protein Design (4 weeks) The culminating exercise is a library/computer project on protein design and engineering. The students propose mutational studies of lysozyme to study stability or mechanism, based on the enzyme’s known structure. Perhaps the most exciting area in protein biochemistry today is protein design and engineering. This is an appropriate final project, since by then the students have an appreciation for the complexities of dealing with proteins and should be capable of planning an extension of the project. The students by this point are familiar with the structure of hen egg-white lysozyme (1). The structure of the corresponding enzyme from bacteriophage T4 has also been established (10) and rational site-directed mutagenesis has been carried out to answer specific questions about the factors that affect protein stability (11). The relationship between the two lysozymes can provide a starting point for the students’ manipulations of the enzyme from hen egg white, especially since there are many amino acids conserved in the two structures
Figure 2. The active site of hen egg-white lysozyme. The backbone structure of lysozyme is illustrated as a ribbon model, with side-chains for tryptophan 62, 63 and 108 as stick models. Atomic coordinates for lysozyme were obtained from the Protein Data Bank. The molecule was displayed and manipulated using Quanta (MSI) on an Iris Indigo Workstation (Silicon Graphics).
(12). Many mutations for the hen enzyme have also been described (13), and students are expected to carry out literature searches beyond the references supplied. The students examine structure–function relationships and decide on changes they would like to make in the lysozyme molecule, based on energy calculations and the scientific literature describing previous mutations in this and similar proteins. Several potential mutation sites are illustrated in Figure 2. The active site of the enzyme is depicted, with side-chains of three tryptophan residues indicated. These residues have been implicated in lysozyme’s mechanism of action, although not in actual catalysis. Tryptophan residues 62 and 63 participate in hydrogen bonding to substrate, and steric hindrance with Trp 108 has been thought to induce conformational strain in the substrate, favoring the transition state (reviewed in ref 13). In order to evaluate the importance of these interactions, students have proposed conservative or radical mutations at these sites. Once they have changed the amino acid residue and allowed the model to reach a new minimum energy, they can measure interatomic distances to determine whether or not a substrate can fit into the active site and/or form hydrogen bonds. A subtle variation on mutation of Trp 108 was a proposal to mutate an adjacent alanine residue, such as to displace the Trp further toward the helix and open up the active site. Other mutations, outside of the active site, have been proposed to change the overall stability of the protein (e.g., Gly to Ala within an α-helix, or loss of a disulfide bond). This exercise illustrates the possibilities of modeling proteins. Because it requires that the students set certain parameters and defaults for structural prediction, it helps them to understand the limitations of modeling. The final paper takes the form of a grant proposal. It presents the purification results, rationale for the desired mutation, relevance to previous studies, an overview of techniques applicable to the study of the mutant, predictions as to the characteristics of the engineered protein, and a plan for its purification. These proposals pass through at least one stage of peer review, and each student gives an oral presentation. This project serves to tie together many concepts learned during both the lecture and laboratory portions of the course. As noted above, we have been able to acquire workstations and software for modeling through funding from
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In the Laboratory
an NSF-ILI grant. However, the research proposal exercise can be modified, depending on facilities available and the goals of a particular course. A literature survey alone yields a wealth of information on this enzyme, and students can design experiments in biochemistry or molecular biology to follow up on the papers they have read. Some recent textbooks are accompanied by diskettes that allow visualization of proteins on computer monitors, although not manipulation of the structures. Computer programs such as Hyperchem (Autodesk, Sausalito CA) and Nanovision (ACS Software) allow display of proteins using PDB coordinates, and students can make decisions about which residues to mutate based on their position in the natural structure or in analogous proteins. Peer review of student work is incorporated into many writing courses (14), as well other courses that require multiple drafts of papers. We have found it to be a useful exercise for students in this laboratory course, both for the short paper and the grant proposal, although some care must be taken that students do not reinforce one another’s misconceptions about scientific writing. There are many advantages to the students to this twostage writing process. Most superficial, but not unimportant, is that it forces them to have their ideas in some written form one week before the final assignment is due. More significantly, it is a part of their professional training. The peer reviews are, in general, full of thoughtful and useful advice to their colleagues. They also learn a good deal from reading other drafts—about styles of writing, effective use of figures and tables, and references that might be useful to their own projects. Feedback from students indicates that they particularly enjoy devising their own experimental plans and that they appreciate the chance to work under the different conditions of small groups and as individuals. Not all of the exercises described here will be appropriate for every undergraduate biochemistry laboratory. However, the principle of projects that take several weeks to develop, beginning with basic concepts and working up to independently designed experiments, can be adapted to many settings. Experimental Details for Each Project
Enzyme Assay The substrate, Micrococcus lysodeikticus, is obtained from Sigma (St. Louis). A suspension (0.3 mg/mL) is prepared fresh in 0.1 M phosphate buffer, pH 6.24, each day. Commercial lysozyme (also from Sigma), 2 mg/mL, as well as a 1:5 dilution of egg white, both in the same phosphate buffer, are used as a source of enzyme. From the change in absorbance over the time of assay, students calculate the activity of each enzyme preparation in terms of units per milliliter. A unit of lysozyme activity is defined as the amount of enzyme that will produce a ∆A450 of 0.001 per min at pH 6.24 at 25 °C. Linearity of the enzyme-catalyzed reaction over the course of 5 min is the criterion used to choose an appropriate dilution of each enzyme preparation. Although the activity of the enzyme is sensitive to effects of pH and ionic strength (15), if these variables are held constant, the assay is quite reproducible and is, in general, very suitable for monitoring purification procedures (16).
Protein Assay Students are supplied with a standard solution of lysozyme at a concentration of 2 mg/mL and the Coo-
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massie blue reagent, obtained from BioRad (Hercules, CA), from which they prepare standards over the range 75 to 1500 mg/mL. Unknowns (in this case, the egg-white solution) are diluted appropriately. A standard curve is prepared by plotting the average absorbance at 595 nm for each set of duplicate readings. Using this standard curve, students determine the protein concentration for each unknown sample.
Independent Assay Project Working in pairs or groups of three, the students design an experiment in which they vary one condition in the enzyme assay for lysozyme or in the Bradford assay for total protein and determine the effects on the assay. Examples of conditions to be varied for the enzyme assay are enzyme concentration, time of incubation, substrate concentration, addition of salts, change in pH or ionic strength of buffer, and addition of sulfhydryl reagent. Examples of conditions to be varied for the protein assay are identity of the protein used as standard (e.g., bovine serum albumin rather than lysozyme), time of incubation, wavelength of measurement, addition of salts, and addition of detergent. In these independent projects, students sometimes discover ways to improve the methodology for the assays, and these improvements are incorporated into the suggested procedures for the rest of the semester. The results of this project are written up as a short paper in a style appropriate for a rapid communication in a scientific journal. The papers go through a round of peer review and revision.
Ion-Exchange Chromatography Once each group has decided on a project and discussed the details with the instructor, they prepare their own buffers and equilibrate the resins. DEAE-Sephacel and CM-Sepharose, both from Pharmacia/LKB (Piscataway, NJ) are supplied, about 20 mL of packed resin for each egg-white preparation. (These resins can be regenerated and reused many times.) Each group uses one egg white for the entire purification project. The egg white is filtered through one layer of cheesecloth and diluted 5-fold with the starting buffer. The first step is done batchwise because of the viscosity of the sample. The diluted egg white is mixed with the resin for approximately 15 min, then centrifuged at 1500 × g for 15 min. The supernatant, containing those proteins not adsorbed to the resin, is decanted. Subsequent washing and elution can then be carried out batchwise or by transferring to a column [1.5 × 20 cm Econocolumns (BioRad)]. The options for recovery of the enzyme from the CM resin include a batch or column method, elution by change in pH or ionic strength, and gradient or stepwise elution. Those carrying out elutions from a column use an automatic fraction collector; gradient formers are available for those who choose to elute with a linear salt gradient. When elution is carried out by a change in pH, it should be noted that lysozyme may precipitate near its isoelectric point, especially at low ionic strength. SDS–polyacrylamide gel electrophoresis is used to monitor purity of the final preparation. Using mini-gels (BioRad Mini-Protean II apparatus), each group can pour, run, and stain their own 8 × 7.3-cm gel within the lab period; destaining can be carried out at any time afterward. The main contaminating band observed is ovalbumin, at a molecular weight of 46,000.
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Computer Modeling Using the program Quanta (MSI, Burlington, MA) on Indigo workstations (Silicon Graphics, Hudson, MA), the students retrieve coordinates from an MSI version of the Protein Data Bank, display the structure, and rationalize what changes would occur with a mutated form of the protein. Even for those who do not have Quanta or analogous programs, structural coordinates are available through the Internet. Students are prepared for their independent use of the molecular modeling workstations through a series of tutorials during the course of the semester. These exercises require that the students become familiar with specific applications of Quanta, including setting secondary conformation and hydrogen bonds, energy calculations, selectively displaying parts of molecules, measuring interatomic distances, and editing existing proteins. This introduction to macromolecular modeling is comparable to that suggested by Harvey and Tan (17) as a brief introduction to the field.
reasoning come more appropriately from the instructor than from peers. The paper is then revised and handed in on the following week. The final draft is accompanied by a summary of revisions and copies of all peer reviews. Students are graded on peer reviews and summaries of revisions and cover letters, as well as on the final paper. Acknowledgments The molecular modeling workstations and associated software were purchased with funding from NSF, Instrumentation and Laboratory Improvement program, grant DUE-9350843 to AJW, with matching funds from Wellesley College. Some of the equipment for protein purification was funded by a grant from the Howard Hughes Medical Institute to Wellesley College. The authors are grateful to Paul Reisberg for helpful discussions in the early stages of this project and to Margaret Merritt for a critical review of the manuscript. Literature Cited
Peer Review For each writing assignment (short paper and grant proposal), one week of lab is devoted to the peer review process. Students are to come to lab with a draft of their paper and a cover letter to their reviewers, which states how far they believe they are in the writing process; what they like and don’t like about their work at this stage; and in what specific areas they need help (e.g., audience level, organization, use of references). They exchange papers, reading two or three during the course of the lab period. For each paper, they fill out a peer review form, which requires that they summarize the paper; look for clarity of presentation, appropriate citations, and use of others’ work; point out any problems in organization or grammar; and suggest the steps necessary to complete the paper. They then go over the peer review form with the writer, referring any questions or disagreements to the instructor. It is most helpful if the instructor can also read and comment on the draft. General comments on the level of detail included and any problems with the
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