Using the protein folding literature to teach biophysical chemistry to undergraduates

Transcription

1 ELSEVIER Biochemistry and Molecular Biology Education 29 (2001) BIOCHEMISTRY and MOLECULAR BIOLOGY EDUCATION Using the protein folding literature to teach biophysical chemistry to undergraduates Spencer Anthony-Cahill **l Department of Chemistty, Western Washington University. Bellingham, WA USA Abstract An understanding of physical chemistry principles enhances student understanding of biochemical phenomena; however, the application of these principles to biological examples is frequently missing in the standard undergraduate physical chemistry curriculum. The topics of protein folding and stability are based in thermodynamics and can serve as a vehicle for presenting essential thermodynamics in a context that is highly relevant to undergraduate biochemistry majors. The outline of a course that replaces the standard thermodynamics offering in physical chemistry is described. The protein folding literature is used to illustrate thermodynamic concepts in this course and students are expected to read and comprehend the assigned literature. The course is offered as a separate biophysical chemistry course for B.S. Biochemistry majors; however, elements of this course may be useful in crafting a morc standard thermodynamics course for B.S. Chemistry majors in chemistry departments seeking to fulfill ACS guidelines for approved B.S. Chemistry majors IUBMB. Published by Elsevier Science Ltd. All rights reserved. The biochemistry major is essentially a double major. Faculty frequently debate the number and kind of courses in biology and chemistry required to satisfy that major while not exceeding credit limits imposed by their institutions. In the context of such constraints it is appropriate to consider the relevance of course material to biochemistry majors, particularly if research is expected of students. In these debates a standard physical chemistry course may be viewed as dispensable because the course content is not generally perceived to be relevant to the study of biomolecular phenomena. Whether or not faculty agree on this point, they should appreciate the fact that undergraduate students will not on their own bridge the gap between the standard physical chemistry curriculum and the application of those physical concepts to biochemical systems. A course that makes explicit the relationships between physical chemistry principles and biological phenomena is arguably more relevant to biochemists. This article describes such a course taught at Western Washington University (WWU). It is an introductory thermodynamics course *Tel.: ; fax: address: sacahill@chem.wwu.edu (S. Anthony-Cahill). /atom.chem.wwu.edu/sacahill/. that employs the protein stability and folding literature to teach the majority of the course content. The academic year at WWU is comprised of three 10 week quarters. This is in contrast to a system of two 14 week semesters common at many colleges and universities. At WWU there is an institutional limit of 110 credits for a major, where one credit is equivalent to 1 h of lecture (or 3 h of laboratory) per week for the 10 week period. WWU graduates B.S. Biochemistry majors each year. Our B.S. Biochemistry degree requires courses in physics, math, chemistry and biology totaling 108 credits (Table 1). These requirements include a full year of physical chemistry. The students take the standard one-quarter offering in quantum mechanics and then switch to a two-quarter sequence of biophysical chemistry. The first quarter of our biophysical chemistry sequence includes fundamental thermodynamics and an introduction to the study of protein stability and folding. The second quarter of the sequence includes mass transport, ligand binding, enzyme kinetics, and spectroscopy (fluorescence, CD, NMR, X-ray crystallography). The basic lesson plan of the protein thermodynamics course fits into a 10 week quarter (Table 2); however, it could be adapted to semesters (see below). We require multivariable calculus, 1 year of introductory calculusbased physics and the first quarter of biochemistry /01/$20.00 :Q 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: SI (0l)OO015-7

2 46 S. Anthony-Cahill / Biochemistry and Molecular Biology Education 29 (2001) Table 1 Recommended schedule of required courses for the B.S. Biochemistry major at WWU Year Fall Winter Spring 1 General Chemistry I/lab Calculus I 2 Organic Chemistry I Intro Biology I Physics I Physics I lab 3 Biochemistry I Analytical Chemistry/ lab Genetics 4 Quantum Mechanics Advanced elective General Chemistry II/lab Calculus I1 Organic Chemistry I1 Organic Chemistry lab Physics I1 Physics I1 lab Biochemistry I1 Biochemistry lab Cell Biology Biophysical Chemistry I General Chemistry III/lab Multivariable Calculus Organic Chemistry 111 Intro Biology I1 Physics 111 Physics 111 lab Molecular Biology Molecular Biology lab Biophysical Chemistry I1 (macromolecular structure and function) as prerequisites. The ability to read and comprehend the literature is an expected outcome for this course. In order to achieve this goal it is necessary to provide students with guidance as they graduate from reading textbooks to reading the primary scientific literature. In the first 3-4 weeks work, heat, the basic thermodynamic state functions and the constant pressure and constant volume heat capacity functions are presented. A standard undergraduate physical chemistry text is a useful resource for the students during this part of the course; however, we use a biophysical text (e.g. [l]) because the end-of-chapter exercises include problems involving biological molecules. In order to prepare students to read the literature some bridging topics are presented in weeks 4-5. These include a discussion of protein denaturation as a two-state phase change described by the Gibbs-Helmholtz equation, determination of a protein stability curve [2], spectroscopic determination of K,, for unfolding (where K,, = [denatured]/[native]) [3,4], the interpretation of differential scanning calorimetry data [5] and the distinctions between calorimetric and van t Hoff analyses [3,5]. This bridging material provides the explicit link between the classical derivations of thermodynamic state functions and the study of biomolecular thermodynamics. Students and faculty may find the online biophysical text available at the Biophysical Society website [6] to be a good resource for some of this material. Weeks 6-8 feature presentations of research articles by the instructor. The instructor illustrates the application of the bridging material to central concepts in protein folding and stability, and demonstrates presentation of the literature by example. This latter point is important since students will present an article to the class in the final weeks of the course. The literature selections currently used in the course at WWU are described in greater detail below. Table 2 Schedule of topics for Biophysical Chemistry I Week Topics Mathematical properties of state functions, thermodynamic analysis of pathways, work, heat AU (AE in some texts), AH, C,, C,, kinetic theory of ideal gases, AH as a function of T, phase changes Protein denaturation as a phase change, statistical and thermodynamic derivations of entropy, calculating AS,,, and AS,,,,, AS as a function of volume and application to folding intermediates Entropy of D vs. N states, Boltzmann distribution function, van t Hoff plots, experimental determination of K,,, Biochemical standard state, intro to calorimetry, van t Hoff vs. calorimetric enthalpies, factors affecting C, in the D and N states, Gibbs-Helmholtz equation, protein stability curves ACp of solvation and the hydrophobic effect, chemical denaturation studies, interpretation of slopes in the transition region, perturbation analysis, entropic and enthalpic effects of mutations on D vs. N states Discussion of Hu et al. [S], application of theory discussed in weeks 1-5 to this paper, Levinthal paradox, folding pathways and intermediate states, the molten globule, amide HID exchange and protection factors Transition state theory, rapid mixing techniques, burst phase analysis, discussion of Jennings and Wright [ll], effects of denaturants on folding and unfolding rates, perturbation analysis to detect structure in the folding transition state Discussion of Matouschek et al. [15,16], calculation of relative energies of intermediates and transition states, kinetic vs. thermodynamic control of folding 9-10 Student presentations The article entitled Contributions of the large hydrophobic amino acids to the stability of staphylococcal nuclease by Shortle et al. [7] provides a good introduction to the determination of the relative stabilities of wild-type and mutant proteins by chemical denaturation methods. This work naturally leads into a discussion of the interpretation of slopes in the transition region and provides a good structural rationale for the experimental

3 S. Anthony-Cahill 1 Biochemistry and Molecular Biology Education 29 (200I) observations described in the paper. In addition, the discussion section is quite provocative in postulating that the denatured state is optimized for maximum structure with maximum exposure of hydrophobic surface. The article Thermodynamics of ribonuclease T1 denatura- tion by Hu et al. [S] introduces an example of calorimetry and compares calorimetric and chemical denaturation data. The derivation of parameters used in the Gibbs-Helmholtz equation and the application of this equation to protein stability problems is clearly illustrated. Differences in the interpretations of calorimetric and van? Hoff enthalpies are also presented. After the analysis of two-state processes is established, the important topic of intermediate states can be introduced into the lecture material. Myoglobin is a protein familiar to biochemistry students and the molten globule state of apomyoglobin has been well studied. The paper Structural characterization of a partly folded apomyoglobin intermediate by Hughson et al. [9] not only introduces the concept of putative folding intermediates but also introduces the use of NMR spectroscopy to determine residual structure in these intermediate states. Good background information on the characteristics of the moltcn globule state can be found in a review by Ptitsyn [lo]. The molten globule described for apomyoglobin was later shown to be on the folding pathway by Jennings and Wright [l 11. Their paper, Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin, extends the work of Hughson et al., and leads into the topics of energy landscapes [12] and folding kinetics [13] very nicely. The effects of denaturants and/or mutations [14] on folding and refolding rates can now be discussed and energy diagrams for folding pathways derived. Two papers by Matouschek et al. [15,16], Mapping the transition state and pathway of protein folding by protein engineering and Transient folding intermediates characterized by protein engineering, illustrate the use of perturbation analysis to define regions of a protein that are structured in the rate-determining transition state. The methods used in this work can be contrasted to the direct detection of structured amide protons in the apomyoglobin intermediate by Jennings and Wright. At this point in the course students can appreciate the possibility that the native state is not necessarily the most thermodynamically stable state on a folding pathway. The article Unfolding conformations of a-lytic protease are more stable than its native state by Soh1 et al. [17] illustrates this concept clearly. The oral presentation assignment is a group project (three students per group) for which the students are expected to develop and deliver a min lecture. Students are encouraged to select an article on their own. Some prefer to select a topic from a list of suitable articles provided by the instructor (see e.g. a list maintained in [IS]). Guidelines for preparing the presentation and a grading scheme are available at the course web site [19]. In general, students indicate that the exercise of preparing a presentation is very important in developing their understanding of the course material, and working in groups is perceived as a benefit. The entire group takes part in the presentation and in answering questions. Six to seven lectures are devoted to this part of the course. The student s comprehension of the articles presented by their peers is tested with a take-home exam. This course is designed to achieve two major objectives: (1) to make the relationship between classical thermodynamics and the study of proteins clear, and (2) to make the literature in this area more accessible to the students. Based on student course evaluations it is evident that most students (but not all) appreciate the literature reading requirement. Some students struggle with a switch from the textbook format and the absence of end-of-chapter problems. Problem sets based on the literature reading assignments need to be made each year and this is a lot of work for faculty. For students involved in research projects a formal requirement to read and evaluate the literature is beneficial to their research efforts. In fact, research students are often motivated to present a paper that focuses on their experimental system, or a technique that they may use to characterize a protein in their lab. Teaching a course like the one described above is not without cost. Splitting the students into physical chemistry and biophysical chemistry tracks requires more faculty resources; however, classes are also smaller (in this case roughly 20 students). It is not necessary to offer a completely separate yearlong track of biophysical chemistry. Our program splits the B.S. biochemists and chemists after one quarter. Similarly for a semester system, one can imagine teaching both groups of students quantum mechanics and basic thermodynamics during the first semester and then moving the biochemists into a second semester of biophysical topics that begins with the material included in the last 7 weeks of the course described above. We think that the biophysical course described above provides B.S. Biochemistry students with a solid background in the introductory thermodynamics one needs to understand a significant portion of the biochemical literature and thereby better serves their needs than does the standard offering in physical chemistry. Not all biochemistry/molecular biology programs have the resources to offer separate courses in biophysics; however, for those faculty desiring more physical chemistry in the curriculum, certain elements of the course described above can be incorporated into the yearlong biochemistry sequence. For example, most biochemistry texts describe the thermodynamics of protein stability in terms of the equations: AG =AH - TAS

4 48 S. Anthony-Cahill / Biochemistry and Molecular Biology Education 29 (2001) and AG = - RT In K,, (where K,, = [denatured]/[native]). The latter equation is used for the determination of the unfolding free energy change, AGOHzO, from equilibrium unfolding studies [3,4]. The methods and theory used for determination of [denatured]/[native] from spectroscopic data and the calculation of by extrapolation [3] or linear regression [4] can be presented in 1-2 lecture hours. If so desired, another lecture can be devoted to the interpretation of the cooperativity of the unfolding transition and its relation to the hydrophobic effect [7]. Relatively short articles that describe the application of these methods, suitable for biochemistry students, can be found in the protein folding literature to supplement textbook reading assignments (see e.g. [20]). It is recommended that the application of the Gibbs-Helmholtz equation to protein stability is presented in a physical chemistry course (see below) rather than in a biochemistry course. This will allow a thorough derivation of the thermodynamic state functions (which are part of the physical chemistry curriculum) without consuming a lot of lecture time in the biochemistry course. The American Chemical Society (ACS) has recently increased the amount of biochemistry required for an approved B.S. Chemistry program. Chemistry departments can fulfill this requirement by requiring one or more biochemistry courses, and/or by incorporating more biochemistry into other areas of the chemistry curriculum. Analytical and organic chemistry courses are obvious choices when considering where in the chemistry curriculum to include more biochemical examples; however, there are a few topics covered in the biophysical course described above that could be included in a more standard physical chemistry course. Protein denaturation can be presented as a phase change described by the Gibbs-Helmholtz equation [2,21], as can changes in entropy with increasing volume as a protein unfolds from a compact native state to an expanded denatured state. A detailed description of the hydrophobic effect [22,23] and increased heat capacity [S] for unfolded proteins would also be appropriate for a rigorous thermodynamics course. All three of these topics require only a superficial understanding of protein folding and structure and therefore should be relatively easily incorporated into a chemical thermodynamics course. The focus of this article is protein thermodynamics; however, other parts of a standard physical chemistry curriculum can also be revised to include biochemical examples. Isotope effects [24] and hydrogen tunneling [I251 in proteins can be presented as part of a quantum mechanics course with very superficial understanding of protein structure (for the purpose of this discussion proteins are simply very large organic molecules). The topic of chemical kinetics can be expanded to include biochemical reactions ranging from simple pseudo-first order reactions (e.g. O2 binding to myoglobin) to enzyme-catalyzed reactions of significantly greater complexity [26,27]. In kinetics the focus of the discussion is rates and rate expressions and not necessarily the structure of catalysts, thus the details of enzyme structure can be downplayed. It is likely to require substantial faculty effort to revise physical chemistry courses to include more biochemistry, in spite of the fact that only superficial knowledge of protein structure and function would be required of the students and faculty. This is in large part due to the inevitable communication difficulties that arise between technical disciplines. Physical chemistry and biochemistry each have distinctive vocabularies that make the literature in each field dense to the uninitiated. In addition, most standard physical chemistry texts include little or no biophysical chemistry, and many biophysics texts presuppose knowledge of basic biochemistry (and in some cases lack the mathematical rigor of.the standard texts). To help overcome these problems, two sources cited herein [6,26] are particularly recommended to physical chemists seeking to incorporate biochemical examples into the physical chemistry curriculum. In the next few years as chemistry faculty respond to the ACS biochemistry requirements, the task of revising chemistry courses may be eased by the appearance of textbooks that include some of the material described above. The inclusion of more relevant physical chemistry into the biochemistry curriculum will enhance student s abilities to grapple with the technical literature and to meet the next challenges of the biotechnology revolution [28]. Whether this is achieved by revising physical chemistry courses or by offering separate biophysics courses will depend on institutional resources. In either case, the students will benefit from a deeper quantitative understanding of their chosen discipline. References c11 I. Tinoco Jr., K. Sauer, J.C. Wang, Physical Chemistry: Principles and Applications in Biological Sciences, 3rd edition, Prentice- Hall, NJ, c21 W.J. Becktel, J.A. Schellman, Biopolymers 26 (1987) * c31 C.N. Pace, Methods Enzymol. 131 (1986) c41 M.M. Santoro, D.W. Bolen, Biochemistry 27 (1988) c51 E. Freire, in: B.A. Shirley (Ed.), Methods in Molecular Biology, Vol. 40, Protein Stability and Folding: Theory and Practice, Humana Press, Totowa, 1995, pp C6l in particular see the chapter by V. Bloomfield at bioph ys/oltb/thermo/part 1-Thermo.pdf. c71 D. Shortle, W.E. Stites, A.K. Meeker, Biochemistry 29 (1990)

5 S. Anthony-Cahill / Biochemistry and Molecular Biology Education 29 (2001) [XI C.-Q. Hu, J.M. Sturtevant, J.A. Thomson, R.E. Erickson, C.N. Pace, Biochemistry 32 (1992) [9] F.M. Hughson, P.E. Wright, R.L. Baldwin, Science 249 (1990) [lo] O.B. Ptitsyn, in: T.E. Creighton (Ed.), Protein Folding, W.H. Freeman and Co., New York, 1992, pp [ll] P.A. Jennings, P.E. Wright, Science 262 (1993) [12] K.A. Dill. H.S. Chan, Nature Struct. Biol. 4 (1997) [I31 F.X. Schmid, in: T.E. Creighton (Ed.), Protein Folding, W.H. Freeman and Co., New York, 1992, pp [14] D.P. Goldenberg, in: T.E. Creighton (Ed.), Protein Folding, W.H. Frccman and Co., New York, 1992, pp [15] A. Matouschek, J.T. Kellis Jr., L. Serrano, A.R. Fersht, Nature 340 (1989) [16] A. Matouschek, J.T. Kellis Jr., L. Serrano, M. Bycroft, A.R. Fersht, Nature 346 (1990) [17] J.L. Sohl, S.S. Jaswal, D.A. Agard, Nature 395 (1998) [18] M. Blaber at: l.sb.fsu.edu/bch5887/literaturelist/literature-list.htm1. [19] [20] N.Y. Protasova, M.L. Kireeva, N.V. Murzina, A.G. Murzin, V.N. Uversky, 0.1. Gryaznova, A.T. Gudkov, Protein Eng. 7 (1994) [2l] P.L. Privalov, in: T.E. Creighton (Ed.), Protein Folding, W.H. Freeman and Co., New York, 1992, pp [22] W. Kauzmann, Adv. Protein Chem. 14 (1959) [23] K.P. Murphy, in: B.A. Shirley (Ed.), Methods in Molecular Biology, Vol. 40, Protein Stability and Folding: Theory and Practice, Humana Press, Totowa, 1995, pp [24] J. Suhnel, R.L. Schowen, in: P.F. Cook (Ed.), Enzyme Mechanism from Isotope Effects, CRC Press, Boca Raton, FL, 1991, pp [25] J.P. Klinman, in: P.F. Cook (Ed.), Enzyme Mechanism from Isotope Effects, CRC Press, Boca Raton, FL, 1991, pp [26] A.R. Fersht, Structure and Mechanism in Protein Science, W.H. Freeman and Co., New York, 1998, pp [27] I.H. Segel, Enzyme Kinetics, Wiley, New York, [28] K.A. Dill, Nature 400 (1999)