In this special online-only article, Ben Caldwell explains how he uses a Journal of Biological Chemistry paper for a more in depth examination of trimeric G-proteins in a biochemistry II course.
|Figure 1 clearly demonstrates the different elution profiles of the two proteins.
Reading primary literature is one of the essential skills undergraduate students need to learn and master. This takes a great deal of practice and certainly does not happen overnight. Unfamiliar jargon and methodologies can be frustrating for students, making finding journal articles at an undergraduate reading level challenging for many instructors. The unfortunate truth is that most scientific research articles simply are not written for novice readers. Identifying an appropriate article that is well written, readable AND content-rich for undergraduates is challenging.
For novice readers, articles need to be written clearly. (On the other hand, reading poorly written paper can also serve as a good example of how not to write). A reader needs to consider a number of questions while reading any research paper, and students need to build an awareness of these issues in addition to being knowledgeable about vocabulary, jargon and applicable methodologies. Students should be given this list of questions to consider:
1) What is the purpose of the study or the questions being examined?
2) What are the conclusions?
3) What are the bases for the conclusions, and are they supported by the experimental evidence?
4) Are the experiments presented in the article applicable to the question(s) being examined?
5) Are the conclusions reasonable based on the evidence or data?
One of the papers I particularly like to use is “Fluoride Activation of the Rho Family GTP-binding Protein Cdc-42Hs” by G. Hoffman et al. We use this article for a more in depth examination of trimeric G-proteins in our biochemistry II course. Although the work described in the paper is not current by today’s publishing standards, G-proteins are an important area of ongoing research, and this paper provides a good example of a well done study of the factors required for association of the alpha-subunit of Rho family GTPases with their corresponding GTPase-activating proteins. More importantly, it is well written and relatively easy for students to read and comprehend. The article effectively demonstrates to students how a variety of seemingly unrelated experimental methods can be used in a complementary manner to provide supporting evidence. I like this particular paper because it presents the research questions methodically, and progressively moves through the data in a manner students can follow.
It should be noted that I provide the article to students initially without the abstract or the discussion sections so as not to provide the students with the authors' final conclusions. When BeF3-1 enters the paper there is usually a great deal of conversation about why the authors included this variation. Examination of the crystal structures of myosin and comparison of the binding data generated by the fluorescence anisotropy experiments usually leads to lively discussions about whether the BeF3-1 or AlF4-1 is a more true analogy of GTP in the active site. We often take three to four class periods to dissect the experiments, discuss the data and examine the crystal structures. By the time we are done, students generally have a good understanding of G-proteins and their co-factors and targets, as well as having reviewed some known methods and learned a few new techniques.
|Figure 2 follows with supporting data using SDS-PAGE analysis of the complexed mixture collected from the gel filtration process.
The introductory section of the paper provides a good starting point for students, describing heterotrimeric G-proteins, contrasting their make-up to smaller monomeric Ras-like GTPases and explaining the similar structural features of the catalytic domains of the two types of G-proteins. Normally, students in my course already have spent time in class examining the different types of G-proteins, so this provides a familiar basis for students with which to begin as a key feature of the study is the use of GTP analogs. The authors introduce the concept of combining two separate chemicals to emulate a single molecule; utilization of GDP in the presence of AlF4-1 to emulate GTP and “activate” the trimeric G-protein.
The study uses a variety of methods which are clearly explained in the experimental procedures section, including protein expression and purification of His-tagged proteins and three different methods to demonstrate association of the two main proteins: gel filtration chromatography, 19F NMR and fluorescence anisotropy. Gel filtration is used to examine the association of the Cdc42 and Cdc42-GAP proteins, and Figure 1 clearly demonstrates the different elution profiles of the two proteins and how, in the presence of GDP and AlF4-1, the two form a larger complex that elutes earlier than the individual components.
Figure 2 follows with supporting data using SDS-PAGE analysis of the complexed mixture collected from the gel filtration process. Note that the figure shows students that the complexation of the Cdc42 and its GAP is not 100 percent efficient, and that some of the individual proteins did not associate together and elute from the column in their original monomeric form. This is initially surprising to the students since they often assume that reactions like this go to 100 percent completion. The two figures complement one another nicely.
|19 F NMR is used to confirm binding of the AlF4-1 to Cdc42 .
19 F NMR is used to confirm binding of the AlF4-1 to Cdc42 (Figure 3). Again, the panel showing the NMR data is well organized and easy for students to follow. The fluorine peak shows no splitting patterns, since all four fluorines are equivalent. This is a good opportunity to reinforce students' previous exposure to NMR in their organic chemistry laboratory courses, where most of their work has been with 1H NMR spectroscopy. Binding of AlF4-1 to Cdc42 in the presence of GDP and Cdc42-GAP is supported by the emergence of a new 19F peak only in the presence of all of the required factors. In the absence of GDP or the Cdc42-GAP a single 19F peak represents AlF4-1 that is free in solution. The emergent upfield peak observed only in the presence of GDP and Cdc42GAP represents the AlF4-1 that is bound to the Cdc42-Cdc42-GAP complex. The upfield chemical shift is a result of the fluorines being in a different, isolated chemical environment in comparison to the AlF4-1 that remain free in solution. Again, this is a good opportunity to discuss and reinforce what students have already learned about NMR and different chemical environments.
Hoffman and coworkers used AlF4-1 but also utilized a second gamma-phosphate mimic in the form of BeF3-1 which has been used in previous studies with myosin in combination with ADP as an analog for ATP (1, 2). I have the students examine the crystal structures and compare the geometries of AlF4-1 and BeF3-1 in the active site of myosin with ADP to observe that BeF3-1 represents takes on a geometry more similar to the gamma-phosphate of ATP, making it a more true phosphate analog.
The final results section of the paper examines the relative affinity of Cdc42 and Cdc42-GAP under varying conditions using fluorescence anisotropy. This is not a technique with which most of our students are familiar, presenting an opportunity to introduce a completely new technique, which also makes this the most challenging section of the paper for the students to grasp. Once we have discussed the basic theory of fluorescence and fluorescence anisotropy (see reference 3 to help introduce this topic), we begin examining the final figures of the paper. The data provide a good steady progression to introduce the method in general and then moves methodically to examine the factors influencing the complexation of CDc42 and its GAP.
This article is an example of a good article to share with students to help them gain experience and confidence with reading primary literature while learning new theory and developing critical thinking skills.
1. Sternweis, P. C., and Gilman, A.G. (1982) Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc. Natl. Acad. Sci. U.S.A. 79, 4888-4891.
2. Fisher, A.J. , Smith, C.A., Thoden, J.B., Smith, R., Sutoh, K, Holden, M., Rayment I. J. (1995) X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. Biochemistry 34, 8960-8972.
3. Royer, C. (1995) Approaches to Teaching Fluorescence Spectroscopy. Biophysical J. 68, 1191-1195.
Ben Caldwell (firstname.lastname@example.org) is a professor of chemistry and chairman of the department of chemistry at Missouri Western State University. He also is a regional director of the Undergraduate Affiliate Network.