Initial Publication Date: August 22, 2021 | Reviewed: March 11, 2023

Analysis of the effects of protein-protein interactions on signaling through a team-based undergraduate biochemistry laboratory course

Daniela Fera, Swarthmore College
Location: Pennsylvania

Abstract

We developed a research-based laboratory course centered on a biological problem involving the B-Raf kinase, specifically the mutant that is commonly found in melanomas. One of the major goals of the project for the students is to generate mutants to determine whether a particular region of the B-Raf protein is critical for the interaction with MEK kinase, a downstream target in the pathway.

Students analyze the published B-Raf-MEK crystal structure and choose a mutation to generate in B-Raf or MEK that might alter the dissociation constant (KD) of the complex. They design primers, perform PCR to generate their desired mutant, transform and purify the resulting DNA, express the DNA in E. coli, and purify the protein, all before characterizing it. Characterizing the mutant proteins consists of performing basic pull-downs, western blots, spectroscopic absorbance assays, and biolayer interferometry for binding kinetics. Students also engage in group meeting presentations and journal clubs in which they discuss their work and related primary literature, respectively. Group meeting and journal club discussions provide a forum for students to come up with new ideas to analyze their results, or for future work. Students summarize key results in a final presentation and paper, and develop a research proposal based on their work.

Data that students obtain from their mutants provide evidence of the importance of a binding region for B-Raf-MEK complex formation, as well as downstream phosphorylation events. Such data will inform future drug discovery programs, as well as form the foundation for students' work in the course the following year. Because working with mutants can result in unpredictable data and results, students sometimes have to adjust their protocols and repeat experiments. Thus, the CURE format of this course also gives students an opportunity to learn to troubleshoot when things do not work as expected, which helps them learn resiliency in science.

Student Goals

  1. Gain experimental and computational skills.
  2. Communicate scientific findings and ideas with classmates and scientists.
  3. Contribute to science via the production of data and through publications.

Research Goals

  1. Identify an allosteric site that is important for protein-protein interactions and can potentially act as a drug target.
  2. Determine the effects of downstream phosphorylation activities of kinases by altering interactions of protein kinases upstream in a signaling pathway.

Context

Only 8-12 students in their junior or senior year enroll in Biochemistry II and the Advanced Experimental Biochemistry laboratory course (the CURE), most of whom are biochemistry majors, though sometimes biology and chemistry majors also enroll. Biochemistry I is a pre-requisite for the course. In Biochemistry I laboratory sessions, students build necessary basic skills in pipetting, and are introduced to buffer preparation, protein expression and purification, and some scientific writing. The experiments do not follow a research-based format and the same experiments on the same systems are offered yearly. In the CURE, students work in pairs and carry out a research project in which they design and generate their own protein mutant prior to characterizing it using a variety of biochemical experiments.

The CURE is traditionally 13 weeks long, with ~1-2 weeks built in for experiment repeats. The laboratory sections have met twice a week for 3 hours and 15 minutes for each session, though the course is being revised so that students meet once a week for lab for 3 hours and 15 minutes and then 3 times a week for 50 minutes each for either group meetings, lectures, journal club meetings or short experimental setups. The instructional team for each section includes the professor, one laboratory instructor, and one undergraduate teaching assistant.

Target Audience:Major, Non-major, Upper Division
CURE Duration:A full term, Multiple terms

CURE Design

In the mitogen-activated protein kinase (MAPK) signaling pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs, drugs that target allosteric sites are needed. The research in this CURE relies on many students carrying out independent experiments with their individually designed mutants, while everyone as a whole seeks answers to specific hypotheses tied to a central question. These studies will provide a proof-of-concept for studying other kinases and their complexes, and help us better understand the fundamental principles of signaling.

Because a crystal structure of the B-Raf-MEK complex was published, and because our collaborator had identified a single mutation that altered B-Raf's ability to phosphorylate MEK, the project was structured to focus on a single alpha helix of the B-Raf kinase or its binding partner on MEK. Students are trained to use PyMOL and, by analyzing the kinase complex, they are able to calculate distances between atoms and determine which amino acids might be critical for their interaction. Because we have the DNA that encodes the B-Raf and MEK kinases, students are able to drive the research project forward once they have decided on a particular amino acid to mutate.

Students work in pairs to perform experiments. They are given the tools (plasmids, protocols, reagents, training in instruments, etc.) to generate their mutant DNA and then make and characterize their mutant protein. While the course formally meets twice a week for a total of two 3.5-hour laboratory sessions, students have access to the laboratories and equipment needed to carry out their work outside class time. Flexibility is also built in to the course so that students can overcome unanticipated obstacles and troubleshoot as needed. Additionally, several meetings are in the format of "group meetings" and "journal clubs". During group meetings students orally present their data, communicate their results and obstacles, and provide feedback and suggestions to others. These meetings occur four times throughout the semester while students work through testing their hypotheses, so they also create a sense of unity and teamwork for students, while also helping them improve scientific communication. During our four journal clubs, students discuss primary literature related to the scientific problem. Their review of the literature helps them understand experimental controls, interpret protocols, results, and data, contextualize their own research findings, and propose new experiments.

This research has been a collaboration with Dr. Ronen Marmorstein's lab at the University of Pennsylvania. Mutants generated by the students in the CURE have been given to the Marmorstein lab for kinetic studies that supplemented our studies, and the results have been shared with the students to help them better understand the importance of using multiple assays to eliminate the possibilities of artifacts. Results our students obtained have been communicated with our collaborators. Our collaborators have attended our student's presentations and, when this was not possible, the presentation has been shared via email with our collaborators. The results of the research the students have conducted has influenced organic syntheses and drug discovery efforts conducted by our collaborators.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Planning and carrying out investigations
Nature of Research: Basic Research, Informatics/Computational Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Identify an allosteric site that is important for protein-protein interactions and can potentially act as a drug target.
Research Goal 2: Determine the effects of downstream phosphorylation activities of kinases by altering interactions of protein kinases upstream in a signaling pathway.


Student Goal 1: Gain experimental and computational skills.

- Practice using scientific 3D visualization software (PyMOL) for identifying regions of B-Raf that may contact MEK, and generating scientific hypothesis.

- Use web tools to do analyze protein and DNA sequences, and generate mutagenesis primers.

- Carry out DNA recombinant techniques: Using PCR to do site-directed mutagenesis, DNA gel electrophoresis, purifying DNA using miniprep procedure.

- Use E. coli for protein production and various forms of chromatography to obtain pure proteins.

- Gain experience with high level instrumentation, such as the AKTA Pure FPLC for purifying proteins.

- Perform pull downs and biolayer interferometry to analyze binding between two proteins.

- Use software to calculate dissociation constants.

- Compare data with controls to establish an argument.

- Perform phosphorylation assays and western blots to analyze phosphorylation levels.

- Analyze western blots in primary literature to interpret data obtained in the course.

- Use software to quantify band intensities.

- Read and discuss primary literature to better understand the relationship between structure and function and how it applies to our project of changing protein-protein interactions and downstream phosphorylation.

- Compare data with controls and with binding experiments to establish an argument, or determine inconsistencies and the need to repeat certain experiments.



Student Goal 2: Communicate scientific findings and ideas with classmates and scientists.

- Write the methods, results, and conclusions for each experiment similar to what is observed in publications. Obtain feedback that would later be incorporated into group meeting presentation.

- Read and discuss primary literature articles with the class during journal club meetings, and put in the context of the research project being performed.

- Generate publication quality figures and tables of data obtained and compile into a PowerPoint presentation.

- Present at group meetings with team-member in front of class and get feedback.

- Write the methods, results, and conclusions for each experiment similar to what is observed in publications. Obtain feedback that would later be incorporated into group meeting presentation.

- Read and discuss primary literature articles with the class during journal club meetings, and put in the context of the research project being performed.

- Make scientific arguments and conclusions using reasoning based on data obtained in the research project and in papers read.

- Generate publication quality figures and tables of data obtained and compile into a PowerPoint presentation.

- For the final group meeting, present with team-member in front of class, collaborators, and other department members.

- Demonstrate understanding of the data in the Results and Discussion sections of the final research paper and in final group meeting presentation.



Student Goal 3: Contribute to science via the production of data and through publications.

- Perform experiments and collect research data

- Synthesize knowledge and analyze data

- Evaluate data and draw conclusions

- Perform experiments and collect research data

- Synthesize knowledge and analyze data

- Evaluate data and draw conclusions

- Determine if the results support the hypothesis or not: does the mutant affect downstream phosphorylation activity?

- Write and revise research paper and/or publication drafts.

- Determine what remaining questions exists and/or what experiments to do in the future, and incorporate in discussion section of final research paper and/or in research proposal.


Instructional Materials

The protocols and instructional materials for this CURE change yearly, especially as I change the format to one lab meeting per week and as the research the course generates gets published. Please email me at dfera1@swarthmore.edu with questions or for the most recent instructional materials.

Syllabus and Timeline (Zip Archive 14.6MB Aug21 21)
Protocols (Zip Archive 52.5MB Aug21 21)
Supplies and Equipment (Excel 2007 (.xlsx) 17kB Aug21 21)

Assessment

Course objectives are formally assessed using anonymous course-specific evaluations administered mid-semester and at the end of the course. Most project goals are subject to summative assessment. Summative assessments are carried out for each activity (pre- and post-lab). Scientific expertise and communication are evaluated when students present their research progress during group meetings, during their final presentation in front of other faculty and students, and when they discuss primary literature articles during journal club meetings. These meetings occur throughout the semester, every few weeks. Student performance between different meetings are compared to check for changes. Student-produced figures are also required and evaluated to help them improve their understanding of the system under study. Reading and writing assignments are evaluated and are expected to improve scientific writing skills. Students produce drafts for formative assessment, especially for their final paper and manuscript, which require high quality, publication-style materials. Scientific writing skills are subject to summative assessment as well, specifically through a short written proposal at the beginning of the semester, summaries of article readings for journal club meetings, laboratory notebook entries throughout the semester, and a written paper at the end of the semester. Rubrics assess students' overall understanding of chemical principles, experimental design, scientific reasoning, quality of presentation slides and figures, etc. The projects and activities evolve in response to the assessments and evaluations.

Written Assignments (Microsoft Word 2007 (.docx) 149kB Aug21 21)
Lab Notebook Guidelines and Rubric (Zip Archive 182kB Aug21 21)
Group Meeting Presentation Guidelines and Rubric (Zip Archive 176kB Aug21 21)

Instructional Staffing

Prof. Daniela Fera: Course instructor and researcher, curriculum design, course materials development, course implementation, course coordination, guides the research project.

Lab prep instructor: Sets up instruments, orders lab supplies, and assists students in the lab with questions on techniques.

Undergraduate teaching assistant: typically a course alum or research student in the Fera research laboratory; assists students in the lab with questions on techniques and computer programs.

Ronen Marmorstein: Scientific Collaborator

 

Author Experience

Daniela Fera, Swarthmore College

The scientific method involves developing hypotheses, carrying out experiments, and making observations. Data that are obtained are analyzed and interpreted to draw conclusions and can lead to new insights. Biochemistry course-based undergraduate research experiences (CUREs) offer a means of practicing these approaches in the classroom. Thus, we developed a research-based laboratory course centered on a biological problem in which students can work on individual projects while contributing to a broader research question as a team.


Advice for Implementation

The CURE has several components: a laboratory section in which computational or experimental work is done, journal club meetings in which students meet to discuss primary literature articles related to the research project being conducted, and group meetings in which students present their data to the class using PowerPoint.

During the previous two times I taught the course, it was sometimes difficult to run a journal club or group meeting the same time an experiment had to be done. My approach to this problem was to change the course format so that there is only one lab session a week and three 50-minute sessions and the journal clubs and group meetings could take place during the latter sessions.

Group meetings: During these meetings, students present their work. Since it can be repetitive to hear all students give a similar complete "background" to the project, it is suggested to them to only provide information relevant to their particular mutation and describe only the most recent results since their last presentation. For the first one or two group meetings, it would helpful to give students some guidance on how to organize their presentations and what information to include or not include. It may also help to provide an example group meeting presentation from a previous year.

Journal clubs: The first paper is assigned very early in the semester and is key to students generating their hypotheses. Since many students at this stage of their college careers have not yet taken an upper level seminar in which they read the primary literature, it can be challenging for them to read and discuss an entire paper without some guidance. To help them, I have them each come up with a question for the class and I compile these questions and distribute all of them a few days before our journal club meeting. Students are asked to try to answer all the questions on their own before the meeting and be prepared to discuss. Additionally, I provide a form they should fill out that helps them think about certain aspects about the paper they are reading (see attached "Journal Club Form" above). During the second iteration of this CURE, I also found it helpful to have students focus on only several figures of the first paper and the related text, particularly figures 2, 3, 5, and 7 of the Haling et al paper.

Labs: One of the challenges with the CURE is that there are a lot of experiments to be done. It helps to keep the pre-lab lectures short (like 5-10 minutes), and spend more time discussing background during different meetings. Another recommendation is to take out one or two of the characterization experiments to allow more time for extra software practice, experiment repeats and/or writing workshops.

At the beginning of the semester, students need to use PyMOL to come up with a mutation they would like to make. While they have used PyMOL in Biochemistry I lab, they have only used it once and so they are not well versed in it. It helps to have them practice with it for more than one or two sessions so that they have a better handle on it before the computational assignments in the CURE.

Since some of the experiments require students to come to lab outside of the regularly scheduled class time, they are given some lab sessions "off" to compensate for this. Additionally, during some of the later sessions, students found it difficult when they were repeating experiments and doing a second experiment at the same time. Once students learned to "divide and conquer" they were fine with it. Removing one or two of the characterization experiments may also allow for more time to minimize this challenge.

For the research paper related to the project they performed, students complete various sections (methods, results, and discussion) as they go through the various experiments and get feedback. As they repeat experiments, they can revise their written work. These all get compiled into their final paper. Students found in beneficial to have this extra (outside of lab) work throughout the semester, rather than having it all done at the end. For their proposal, however, it would have been helpful to hold a few writing workshops, detailing things such as formats for such a paper, peer review, etc. Because of how many experiments were in the course, we did not get a chance to do this before. The course is now being updated to allow for more writing workshops, so please feel free to contact me to find out how it went.


SUPPLIES: DNA constructs for B-Raf kinase domain, full length His-MEK, and GST-ERK. Please see spreadsheet "Supplies and Equipment" above for a more detailed list of supplies with some ordering information. Each protocol also indicates supplies and equipment needed.

EQUIPMENT: It would help to have a cold room, especially for the protein purifications. Micropipettors are also essential. Additional equipment is listed in the spreadsheet above "Supplies and Equipment". Each protocol also indicates equipment needed.

SOFTWARE: PyMOL (free), ImageJ (free), GraphPad Prism (requires license)

Iteration

Student reflections on "failed" experiments take place via written assignments, as well as during our group meetings, of which there are four throughout the semester and have taken place during lab time. Flexibility is built into the course so that students can overcome unanticipated obstacles and troubleshoot as needed. Specifically, there are at least 1-2 weeks of lab sessions in which no experiments are scheduled, but can be used for experimental repeats. Students can also sometimes take care of certain aspects of their experiments outside of class time.

The course can essentially be broken into three stages that come together to form the larger research project. First, after each type of experiment performed, students are asked to write a "methods, results, and discussion" section. In this write-up, they are asked to reflect on what may have gone wrong (if something did not work or if the data did not look good), and what changes could be useful going forward. They receive feedback with suggestions on how to proceed. Sometimes this entails repeating the same experiment all over again; other times it entails making adjustments to their previous experiment, such as by changing concentrations of the proteins they used. Then, after each stage, students present their work to the group using PowerPoint, allowing for further feedback on how to proceed.

There are a few points at which students can repeat their experiments or revise their hypothesis. The first comes in the first stage when students use scientific programs to visualize available macromolecular structures and analyze sequences to generate their own hypotheses. We meet as a group to discuss, and students are permitted to make changes at this point, which will set the stage for their project for the rest of the semester. Then, in the second stage, students produce reagents to test their hypotheses, i.e. by constructing plasmids, and expressing and purifying proteins. During the mutagenesis process, students may find that they have no bacterial colonies to work with or their sequenced DNA does not contain the mutation of interest. Students are given an opportunity to repeat, either by picking new colonies, doing new transformations, etc. Then, in the final stage, after making these materials and their protein mutants, students conduct biochemical assays and analyze results. At this stage, they are given the opportunity to troubleshoot unexpected results and repeat certain experiments to get better data.

Using CURE Data

The course has a Google Drive folder set up for all students to upload all their raw data, analyzed data, and written methods, results and discussions on each piece of data. This work is divided into subfolders based on the kind of experiment done. Quality is ensured by having students run experiments in multiple replicates. Data are presented to the class during group meetings and students are given an opportunity afterwards to repeat their experiments if needed to obtain better quality data.

I share results from the previous CURE with students the following year to provide them with some background information that can be used when they choose their own mutant to make, as well as to get them excited about the project. I share progress and results with collaborators from Ronen Marmorstein's lab (at the University of Pennsylvania) via email. Because of the pandemic, students have not been able to attend conferences to present this work as planned. Instead, I have given oral presentations on this work at virtual conferences and other seminar series and acknowledged all the students that contributed to the research in the CURE.

The research produced from the CURE eventually led to a publication. All students are asked to produce publication quality tables, figures, and written work by the end of the semester. These eventually are compiled into a publication and all students are asked to contribute to the revision process. Students are listed as co-authors in an order that corresponds to their input. For example, a student who continued a research project in my research laboratory after taking the course was listed as first author.

Resources

References for instructors:
1. Johnson LN, Noble ME, Owen DJ. Active and inactive protein kinases: structural basis for regulation. Cell. 1996 Apr 19;85(2):149-58. doi: 10.1016/s0092-8674(00)81092-2. PMID: 8612268.
2. Shaw AS, Kornev AP, Hu J, Ahuja LG, Taylor SS. Kinases and pseudokinases: lessons from RAF. Mol Cell Biol. 2014 May;34(9):1538-46. doi: 10.1128/MCB.00057-14. Epub 2014 Feb 24. PMID: 24567368; PMCID: PMC3993607.
3. Matallanas D, Birtwistle M, Romano D, Zebisch A, Rauch J, von Kriegsheim A, Kolch W. Raf family kinases: old dogs have learned new tricks. Genes Cancer. 2011 Mar;2(3):232-60. doi: 10.1177/1947601911407323. PMID: 21779496; PMCID: PMC3128629.
4. Holderfield M, Deuker MM, McCormick F, McMahon M. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer. 2014 Jul;14(7):455-67. doi: 10.1038/nrc3760. PMID: 24957944; PMCID: PMC4250230.
5. Ascierto PA, Kirkwood JM, Grob JJ, Simeone E, Grimaldi AM, Maio M, Palmieri G, Testori A, Marincola FM, Mozzillo N. The role of BRAF V600 mutation in melanoma. J Transl Med. 2012 Jul 9;10:85. doi: 10.1186/1479-5876-10-85. PMID: 22554099; PMCID: PMC3391993.
6. Jacob R. Haling, et al. (2014) Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling. Cancer Cell. 26: 402–413.
7. Deborah K. Morrison, et al. (1993) Identification of the Major Phosphorylation Sites of the Raf-1 Kinase. Journal of Biological Chemistry. 268(23):17309-17316.
8. Cope, Nicholas et al. (2018) Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein. ChemBioChem. 19:1988-1997.
9. James Tsai, et al. (2008) Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. PNAS. 105(8):3041-3046.
10. Nguyen D, Lin LY, Zhou JO, Kibby E, Sia TW, Tillis TD, Vapuryan N, Xu MR, Potluri R, Shin Y, Erler EA, Bronkema N, Boehlmer DJ, Chung CD, Burkhard C, Zeng SH, Grasso M, Acevedo LA, Marmorstein R, Fera D. Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling. Biochemistry. 2020 Dec 22;59(50):4755-4765. doi: 10.1021/acs.biochem.0c00598. Epub 2020 Dec 3. PMID: 33272017.

References for students:
1. Jacob R. Haling, et al. (2014) Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling. Cancer Cell. 26: 402–413.
2. Deborah K. Morrison, et al. (1993) Identification of the Major Phosphorylation Sites of the Raf-1 Kinase. Journal of Biological Chemistry. 268(23):17309-17316.
3. Cope, Nicholas et al. (2018) Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full-Length Protein. ChemBioChem. 19:1988-1997.
4. James Tsai, et al. (2008) Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. PNAS. 105(8):3041-3046.