Rheostat or Toggle: Examining the role of non‐conserved mutations to LURE students into research

There is a growing movement to involve undergraduate students in authentic research experiences. A variety of studies have indicated the strength of this approach in developing scientific aptitude, confidence, critical thinking skills and likelihood to become career scientists. Course‐based undergraduate research experiences (CUREs) provide the opportunity to lure students into legitimate research at both primarily undergraduate and large research institutions. Here, we describe how to use a CURE‐based biochemistry lab course to explore the role of conserved and non‐conserved amino‐acid sites in structure and function of lactate dehydrogenase enzyme (LDH). Recent research suggests that substitutions at non‐conserved positions behave more like rheostats in modulating protein function, whereas substitutions at conserved positions result in “toggle switching.” In the first phase of this CURE, students used bioinformatics tools to carry out protein sequence alignments and 3D structural explorations to identify potential sites to mutate in the LDH gene. Site‐directed mutagenesis was then used to generate amino acid substitutions at non‐conserved sites in the LDH protein. Students confirmed mutations to the DNA sequence in protein expression vectors before carrying out protein purification of both wildtype and mutant LDH proteins. In the final phase, students assessed protein function using a variety of techniques. All students were required to determine LDH kinetic parameters (K m and K cat ). In addition, students were prompted to explore other enzyme structural or functional changes through alternative assays or broader biophysical and structural studies of their choice. For example, students initiated studies included the use of circular dichroism to determine structural changes and/or comparisons of thermal stability, examination of coenzyme binding effects and conformational changes via fluorescence spectroscopy. Formal assessments were carried out to determine gains in scientific aptitude, confidence and critical thinking. A culminating poster session allowed students to disseminate their research findings beyond the classroom and receive critical feedback from faculty and students from a broader realm. Attitudinal changes resulting from this experience were monitored by administering the CURE assessment tool developed by Lopata et. al before and after the course. Informal attitudinal assessments indicated that participating in this new curriculum increased student interest in attending graduate school and pursuing careers in biochemical research. Student confidence in their ability to conduct independent research also increased. We feel that because the outcome of each experiment was not known beforehand, students were able to primarily focus on the experimental design and data analysis hence increasing their scientific aptitude, confidence and critical thinking skills. Support or Funding Information This research was funded by Wichita State University and McPherson College.