The HICA project

Kathleen Cornely, Robert H Walsh '39 Professor of Chemistry and Biochemistry, Providence College
Katherine Hoffmann, Associate Professor and Chair, John Stauffer Professor of Analytical Chemistry at California Lutheran University
Location: Rhode Island

Abstract

In this CURE, inspired by the work of Hoffmann, et al., students prepare mutant Haemophilus influenzae carbonic anhydrase (HICA) proteins. Using PyMOL to visualize the three-dimensional structure of the HICA protein, students choose one or more surface amino acid residues to mutate to histidine residues in order to create a surface histidine cluster that will allow the mutant protein to bind to a nickel affinity column. Using site-directed mutagenesis, recombinant plasmids are constructed and are then used to transform an E. coli expression vector. The mutant HICA protein is overexpressed, cells are lysed, and students load the cell lysate onto Ni-NTA columns and determine the imidazole concentration required to elute the mutant protein. The construction of a library of mutant proteins will allow the development of a general method in which specific surface histidine residues of any protein can be mutated in order to facilitate affinity purification. The Haemophilus influenzae bacterium described herein is a respiratory pathogen that causes meningitis (in its encapsulated form) and mucosal infections such as otitis media, sinusitis and conjunctivitis (in its unencapsulated form). A recent study showed that the carbonic anhydrase enzyme is absolutely required for pathogenesis. Furthermore, expression of the HICA enzyme allows the pathogen to survive in host immune cells (Langereis, et al.). These observations make the study of HICA itself particularly attractive, in addition to the overall goal of contributing to a body of work that will allow the minimal histidine character required for nickel affinity to be ascertained.

Student Goals

  1. Students build upon their knowledge of protein structure/function relationships covered in the pre-requisite introductory biochemistry course
  2. Students obtain expertise in the use of laboratory techniques commonly used in biochemistry and molecular biology.
  3. Students effectively communicate their results to the scientific community

Research Goals

  1. To use the technique of site-directed mutagenesis to construct H. influenzae carbonic anhydrase mutant proteins and to purify these proteins using nickel affinity chromatography.
  2. To use the binding characteristics of the mutant proteins to develop an algorithm that will allow us to design a streamlined procedure to purify a wide variety of proteins.

Context

This full semester CURE is carried out in the CHM 310L Biochemistry Laboratory course at Providence College. The laboratory course is a three-credit course, meeting twice a week for 50 minutes for lecture and once a week for four hours in the laboratory. The course is required for biochemistry majors, who elect to take the course either their junior or senior year. The course is always offered in the spring. Depending on when the student takes the course, the students have had, at minimum, the first semester biochemistry lecture course (which covers structure/function relationships of macromolecules and an overview of intermediary metabolism) as well as an introductory course in cellular and molecular biology. Some of the students have also taken microbiology and genetics, which both have a laboratory component. Finally, most if not all of the students in the course are participating in independent research, as we have a strong tradition of independent research in our department. The biochemistry laboratory space is small and accommodates eight students. Depending on enrollment, we might run one or two sections each spring.

Target Audience:Major, Upper Division
CURE Duration:A full term

CURE Design

The inspiration for this CURE is based on a study carried out by Hoffmann, et al. (Anal. Biochem. 458:66-28, 2014). Noting that there are several E. coli proteins (including carbonic anhydrase, or ECCA) that have endogenous affinity—in the absence of a terminal histidine tag—to a nickel column, the research team used site-directed mutagenesis to engineer a similar degree of affinity in the Haemophilus influenza carbonic anhydrase protein (HICA, a beta-carbonic anhydrase, which does not have endogenous affinity for nickel), in which a surface arginine was mutated to a histidine. This completed a likely dyad cluster with another nearby histidine and resulted in a protein that mimicked the endogenous affinity of ECCA to a nickel affinity column, without the use of an N- or C-terminal histidine tag. Although the use of such tags for the purposes of purifying proteins using nickel affinity chromatography is commonplace, the possibility exists that such tags might alter protein structure and function and potentially interfere with subsequent experiments. Creating surface histidine dyads in a way that does not interfere with enzymatic activity could provide a viable alternative for a wide variety of proteins other than the carbonic anhydrases investigated here.

At this time, several questions remain unanswered, which are the focus of the CURE: Does the distance between histidine residues influence affinity to a nickel column? How many residues are required for efficient purification?

To answer this question, students use the molecular visualization program PyMOL to identify candidate surface amino acid residues that could potentially form the appropriate histidine dyads that would endow the HICA protein with enhanced affinity to a nickel column. Construction of a library of HICA mutants will allow the development of an algorithm in which the parameters of protein binding to immobilized nickel are clearly defined. In this way, the method can be extended to the purification of other bacterial proteins of interest. Contributions to the project are significant, as a class of students can produce a greater number of mutant proteins than an individual researcher. Students are invested in the project, as they design their own mutant proteins and maintain ownership over the project until its completion at the semester's end.

The biochemistry laboratory course meets the requirement for the Writing Intensive II Proficiency of Providence College's Core Curriculum. There are several objectives that must be met, including (1) the requirement for a variety of writing assignments, (2) the opportunity for ample practice in writing outlines, revising drafts, and editing, (3) the development of student ability to write insightful and well-organized essays, (4) the development of student ability to use various stylistic techniques, and (5) the requirement for the proper use and correct citation of sources.

In this course, we meet the requirements by focusing on each one of the five sections of a scientific paper. We spend class time discussing how the section is to be written (using published papers as models), then each student writes a draft of that particular section. The instructor critiques the drafts and discusses them either with students individually or with the entire class. The drafts are required but are not graded. Students usually write ~five rough drafts prior to submitting the final paper.

Students who complete excellent work are invited by the instructor to publish their final papers on Providence College's Digital Commons, an open-access publishing system for research, scholarship and creative expressions by Providence College faculty and students in all media and formats.Scholarship and creative expressions included within Digital Commons at Providence College are required to be reviewed. Papers published on Digital Commons can be accessed by scholars across the country and around the world.

Students also have the opportunity to share their results with the College community at the Annual Celebration of Student Scholarship and Creativity, held on our campus every spring. The event showcases the scholarly, creative and service work Providence College students engage in on campus, in the community, and around the world.

In addition, students have the opportunity to present their findings at regional meetings, such as the Rhode Island American Chemical Society meeting, or national meetings such as annual meetings of the American Chemical Society and the American Society for Biochemistry and Molecular Biology.

Future employers of students who complete the course can also be identified as stakeholders. In job interviews, students are able to speak to the research skills, bench work and bioinformatics skills obtained in the course, while the instructors are able to provide detailed descriptions of student strengths in their recommendations.

Core Competencies:Analyzing and interpreting data, 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: To use the technique of site-directed mutagenesis to construct H. influenzae carbonic anhydrase mutant proteins and to purify these proteins using nickel affinity chromatography.
Research Goal 2: To use the binding characteristics of the mutant proteins to develop an algorithm that will allow us to design a streamlined procedure to purify a wide variety of proteins.

Student Goal 1: Students build upon their knowledge of protein structure/function relationships covered in the pre-requisite introductory biochemistry course

Using the molecular visualization program PyMOL, the three-dimensional structure of the HICA protein is examined. Surface amino acid residues that are (a) located away from the active site, and (b) reside between 4-7 angstroms away from existing surface histidines are good candidates for mutation. Students will choose surface amino acid residues(s) to mutate to histidine residues, to create histidine surface clusters that do not impact enzymatic activity.

Students perform an analysis of their mutant protein by determining the distances between histidine pairs on the surface of the protein. Mutant proteins that require a high concentration of imidazole to elute them (275 mM or greater) likely have surface histidine pairs that form strong bonds with the immobilized nickel ions on the affinity beads.


Student Goal 2: Students obtain expertise in the use of laboratory techniques commonly used in biochemistry and molecular biology.

Students design their own primers and conduct three rounds of PCR to construct a plasmid expressing the HICA mutant protein of their own design. Following confirmation of the presence of the mutation by Sanger sequencing, students transform their mutant plasmids into a protein expression vector and prepare a cell lysate of their protein that can be used as a starting material for nickel-NTA affinity chromatography. Protein purification is verified by SDS-PAGE.

Students perform an analysis of their mutant protein by determining the concentration of imidazole required to elute them from the column.


Student Goal 3: Students will effectively communicate their results to the scientific community

Students keep a weekly laboratory notebook in which they document their experimental work. Students are provided with instruction in the preparation of an effective laboratory notebook that documents that work in sufficient detail that will allow not just the student, but a successor, to repeat the experiment. The laboratory notebook also serves as a valuable resource that students consult when preparing their journal-style papers, and later, when the work is eventually published.

Students summarize the semester's laboratory work in a journal-style paper in which they discuss their own results and compare them to published results as well as unpublished results from students who have previously participated in the CURE.


Instructional Materials

A detailed syllabus, along with a schedule of experiments, is posted below. In addition, the collection of laboratory protocols distributed to the students is provided below. Finally, I have posted an Appendix that contains the sequence of the pHICA plasmid, a HICA gene translation map, the primer sequences used in the site-directed mutagenesis and sequencing portions of the project, and buffer and media recipes.

Syllabus and schedule of experiments (Acrobat (PDF) 515kB Aug25 21)
Protocols used in the HICA Project (Acrobat (PDF) 2.7MB Aug25 21)
An appendix, containing the sequences of the plasmid, primers used, and recipes (Acrobat (PDF) 368kB Aug25 21)

Assessment

Sample student rubric for laboratory notebook entry (Acrobat (PDF) 75kB Aug25 21)
Rubric/checklist for final paper (Excel 2007 (.xlsx) 16kB Aug25 21)

Instructional Staffing

The CURE is largely the responsibility of the faculty member assigned to teach the course. Valuable assistance is provided by paid undergraduate teaching assistants.

Katherine Hoffmann, associate professor of chemistry at California Lutheran University, was the inspiration for this project and provides a valuable consulting role to the course instructor and students at Providence College

 

Author Experience

Kathleen Cornely, Providence College

Before implementing this CURE, I taught my biochemistry laboratory course in a very traditional way. When I became involved with the SEA PHAGES program I saw that students could benefit from a course-based research experience so I wanted my biochemistry laboratory students to have the same experience.


Advice for Implementation

I have taught four iterations of this CURE. In my experience, it is very important to set the stage for the course on the first day, and to obtain "buy-in" from the students, most of whom have not experienced a CURE previously and may be expecting a traditional laboratory experience. I show students published data (from several of the references below) which convincingly document the advantages of a course run as a CURE. After the first year, I was also able to share success stories with the current students and describe how participation in the CURE benefited their predecessors when they went on the job market or entered graduate school. One student brought along a copy of his research paper to an interview and was subsequently offered the position, another student was offered a position as a lab manager after I was able to provide a lengthy description of the student's strengths in a phone interview. Another student was asked, during her first rotation in graduate school, to carry out a series of tasks very similar to what we do in the CURE, and she was able to undertake those tasks with confidence. Students who fully embrace the concept of a CURE will approach their studies with greater engagement and ownership.

On occasion, students may need to come into the lab after hours to set up a PCR, start cultures, or check plates. At Providence College, students are required to complete a "Research Student: After Hours" form which is approved by the instructor and filed with campus security and Environmental Health and Safety. Framing this procedure as a privilege and referring to the students enrolled in the course as "research students" increases the willingness of the students to perform this work (although the instructor, the student teaching assistant and fellow classmates are always willing to pitch in and help if a student is unable to come to the laboratory outside of class).

I also found that it is important to allay student nervousness about the final assessment, a journal-style research paper that constitutes 50% of the student's grade. Students who have experienced a laboratory course conducted in the traditional way are accustomed to submitting weekly laboratory reports. I have found that scaffolding this assignment by having students complete rough drafts (described elsewhere) in which the instructor provides detailed feedback throughout the course of the semester, allows the students to edit and refine their work and poises the students for success when they complete the assignment at the end of the semester.

Throughout the course, it's important for the instructor to model a growth-mindset approach and encourage the students to do the same, and to frame mistakes as an opportunity to learn.

 

Iteration

The course begins with the students designing their own HICA mutants, using PyMOL as a tool to examine protein structure to search for candidate amino acid residues that might create or complete a surface histidine dyad. Students who took the course after the first year had the advantage of being able to access plasmids (constructed by students in the previous year) as starting reagents to prepare double-, triple- or even quadruple mutants.

The first half of the semester is devoted to constructing the mutant plasmid using site-directed mutagenesis. Proper primer design and good pipetting technique are keys to success. We spend lecture time discussing best practices for PCR primer design. In addition, students are encouraged to consult with other faculty, either the instructor or other faculty members in our department or the Department of Biology who have a high level of skill in primer design. Students are also encouraged to consult technical support services provided by the companies who supply reagents for our project. This normalizes the process of asking for help and assists the students in building their networks.

The site-directed mutagenesis portion of the project involves three rounds of PCR, and good pipetting technique is essential for success. I have several videos on pipetting technique that I show to students in class. We also spend a portion of laboratory time at the beginning of the semester carrying out a simple pipetting calibration exercise to hone and assess this important technique.

During the first week when the students are setting up the first round of their PCRs, it is important, especially if you have a group of students who are very social or who like to listen to music during the laboratory period, that you remove all distractions (no talking, no music) when setting up their first PCR.

Students are involved in trouble-shooting their results, in consultation with the instructor. If any of the PCR reactions are unsuccessful, the student is involved in re-designing the experiment to find the optimal annealing temperature, perhaps by conducting multiple PCRs at different temperatures. Students can also consider the different attributes of "touchdown" versus "classic" PCR when re-designing their experiments.

If one or more PCR reactions are not successful the first time, the experiments can be re-designed, as described above, and repeated. Generally, this involves instructors being willing to help out students and for students to come in on their own time. There are creative ways to accomplish this to minimize the amount of time outside of class for the student. It is not very time-consuming to set up a PCR, and the thermocycler can then run overnight, the sample frozen, and the student can resume the project during the next lab period.

The three rounds of PCR are designed in such a way that students can repeat a procedure with a minimum investment of time. For example, as described in the posted protocols, following the completion of the first round of PCR, the reaction mixture is divided into two lanes of a gel and the students gel-purify both bands to obtain two samples of Megaprimer. If the second round of PCR is unsuccessful, the student can retrieve the second gel-purified sample and re-run the second round of PCR without having to re-run the first round. If the second round of PCR is unsuccessful, the Megaprimer is usually visible on the gel, which the students can gel-purify and then retrieve without having to repeat the first round of PCR.

The second half of the semester, typically following spring break, involves the students carrying out a transformation with their mutant plasmid and over-expressing the protein. The sequencing of the plasmids is accomplished over the break. If the results show that the site-directed mutagenesis experiment was not successful, the students can prepare a culture from a mutant prepared by a student the previous year and perform the transformation and protein expression tasks in the course. Then they can do some research on their own to determine effective ways of encouraging the mutant proteins to bind to a Ni-NTA column.

Over the four years of running this CURE, I have had students construct 20 mutant plasmids that are transformed into DH5-alpha cells. If you are interested in running this CURE, please contact me and I would be willing to share any of these materials with you.

 

Using CURE Data

As a requirement of the course, students write a journal-style research paper that summarizes their accomplishments in the course. This assignment is scaffolded as described above. Students who submit excellent work are invited to apply for The Phillips Memorial Library Undergraduate Craft of Research Prize. In the past four years, one student has won first prize and another student won an honorable mention. Their papers are posted on Providence College's Digital Commons and can be accessed by researchers around the world. In addition, students may post their own papers to our department's Digital Commons site. At the time of this writing, HICA-related papers written by students have nearly 300 downloads.

The instructor has presented the results of this project at two education meetings of the ASBMB and a student presented a virtual poster in the Spring of 2020 at the American Chemical Society meeting on behalf of students involved in the project.

Students involved in the project have constructed twenty single-, double-, triple- and quadruple mutants. We have glycerol stocks of the mutant plasmids in E. coli DH5alpha cells that are available for students at Providence College who wish to pursue independent research projects. We are also happy to share these materials with others interested in the project.

 

Resources

1. Hoffmann (2014) "Surface histidine mutations for the metal affinity purification of a beta-carbonic anhydrase", Anal Biochem 458, 66-68.
2. Cronk, J. D., Rowlett, R. S., Zhang, K. Y. J., Tu, C., Endrizzi, J. A., Lee, J., Gareiss, P. C., and Preiss, J. R. (2006) "Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase", Biochemistry 45, 4351-4361.
3. Langereis, J. D., Zomer, A., Stunnenberg, H. G., Burghout, P., Hermans, P. W. M. "Nontypeable Haemophilus influenzae carbonic anhydrase is important for environmental and intracellular survival", J. Bacteriol., 195, 2737-2746.
4. Rowlett, R. S. (2010) "Structure and mechanism of the beta-carbonic anhydrases. Biochim. Biophys. Acta, 1804, 362-373.
5. Barik, S. (1996) Site-directed mutagenesis in vitro by megaprimer PCR, Methods in Molecular Biology 57, 203-215.
6. Block, H., Maertens, B., Spriestersbach, A., Brinker, N., Kubicek, J., Fabis, R., Labahn, J., and Schäfer, F. Chromatography (IMAC): A Review, Methods in Enzymology, 463, 439-473.
7. Cronk, J. D., Endrizzi, J. A., Cronk, M. R., O'Neill, J. W., and Zhang, K. Y. (2001), "Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity. Protein Sci. 10(5), 911-922.
8. DeLano, W. L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, Palo Alto, CA USA http://www.pymol.org
9. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991) 'Touchdown' PCR to circumvent spurious priming during gene amplification, Nucleic Acids Res. 19 (14), 4008.
10. Kuh, George D. (2008). High-Impact Educational Practices: What they are, who has access to them and why they matter, AAC&U.
11. Supuran, C. (2011) "Bacterial carbonic anhydrases as drug targets: toward novel antibiotics?" Front. Pharmacol. 2, 1-34 (10.3389/fphar.2011.00034).
12. Smith, K. S., and Ferry, G. (2000), "Prokaryotic carbonic anhydrases". FEMS Microbiol. Rev. 24, 335-366.
13. Waterman, R., and Heemstra, J. (eds.) (2018), "Expanding the CURE model: course-based undergraduate research experience", Research Corporation for Science Advancement.