Ellis Bell

Foundational concepts and underlying theories for majors in “biochemistry and molecular biology”

Over the past two years, through an NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members and science educators from around the country that focused on identifying: 1) core principles of biochemistry and molecular biology, 2) essential concepts and underlying theories from physics, chemistry, and mathematics, and 3) foundational skills that undergraduate majors in biochemistry and molecular biology must understand to complete their major coursework. Using information gained from these workshops, as well as from the ASBMB accreditation working group and the NSF Vision and Change report, the Core Concepts working group has developed a consensus list of learning outcomes and objectives based on five foundational concepts (evolution, matter and energy transformation, homeostasis, information flow, and macromolecular structure and function) that represent the expected conceptual knowledge base for undergraduate degrees in biochemistry and molecular biology. This consensus will aid biochemistry and molecular biology educators in the development of assessment tools for the new ASBMB recommended curriculum.

What skills should students of undergraduate biochemistry and molecular biology programs have upon graduation

Biochemistry and molecular biology (BMB) students should demonstrate proficiency in the foundational concepts of the discipline and possess the skills needed to practice as professionals. To ascertain the skills that should be required, groups of BMB educators met in several focused workshops to discuss the expectations with the ultimate goal of clearly articulating the skills required. The results of these discussions highlight the critical importance of experimental, mathematical, and interpersonal skills including collaboration, teamwork, safety, and ethics. The groups also found experimental design, data interpretation and analysiand the ability to communicate findings to diverse audience to be essential skills. To aid in the development of appropriate assessments these skills are grouped into three categories, 1) Process of Science, 2) Communication and Comprehension of Science, and 3) Community of Practice Aspects of Science. Finally, the groups worked to align these competencies with the best practices in both teaching and in skills assessment.

Essential concepts and underlying theories from physics, chemistry, and mathematics for “biochemistry and molecular biology” majors

Over the past two years, through an NSF RCN UBE grant, the ASBMB has held regional workshops for faculty members from around the country. The workshops have focused on developing lists of Core Principles or Foundational Concepts in Biochemistry and Molecular Biology, a list of foundational skills, and foundational concepts from Physics, Chemistry, and Mathematics that all Biochemistry or Molecular Biology majors must understand to complete their major coursework. The allied fields working group created a survey to validate foundational concepts from Physics, Chemistry, and Mathematics identified from participant feedback at various workshops. One‐hundred twenty participants responded to the survey and 68% of the respondents answered yes to the question: “We have identified the following as the core concepts and underlying theories from Physics, Chemistry, and Mathematics that Biochemistry majors or Molecular Biology majors need to understand after they complete their major courses: 1) mechanical concepts from Physics, 2) energy and thermodynamic concepts from Physics, 3) critical concepts of structure from chemistry, 4) critical concepts of reactions from Chemistry, and 5) essential Mathematics. In your opinion, is the above list complete?” Respondents also delineated subcategories they felt should be included in these broad categories. From the results of the survey and this analysis the allied fields working group constructed a consensus list of allied fields concepts, which will help inform Biochemistry and Molecular Biology educators when considering the ASBMB recommended curriculum for Biochemistry or Molecular Biology majors and in the development of appropriate assessment tools to gauge student understanding of how these concepts relate to biochemistry and molecular biology.

Organelle and translocatable forms of glyoxysomal malate dehydrogenase

Many organelle enzymes coded for by nuclear genes have N‐terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p‐gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+–NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p‐gMDH to be dimers in solution with p‐gMDH having a correspondingly higher molecular weight. p‐gMDH also exhibited a smaller translational diffusion coefficient (Dt) at temperatures between 4 and 32u2003°C resulting from the extra amino acids on the N‐terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p‐gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the Km for oxaloacetate. Both the organelle‐precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p‐gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55u2003Å. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50u2003Å. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p‐gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N‐terminal.

Ligand-induced changes in the conformational stability and flexibility of glutamate dehydrogenase and their role in catalysis and regulation

Bovine glutamate dehydrogenase (GDH) is allosterically regulated and requires substrate‐induced subunit interactions for maximum catalytic activity. Steady‐state and presteady‐state kinetics indicate that the rate‐limiting step depends on the nature of the substrate and are likely associated with conformational fluctuations necessary for optimal hydride transfer. Deuterated glutamate shows a steady‐state isotope effect but no effect on the presteady‐state burst rate, demonstrating that conformational effects are rate limiting for hydride transfer while product release is overall rate limiting for glutamate. Guanidine hydrochloride unfolding, heat inactivation, and differential scanning calorimetry demonstrate the effects of alternative substrates, glutamate and norvaline, on conformational stability. Glutamate has little effect on overall stability, whereas norvaline markedly stabilizes the protein. Limited proteolysis demonstrates that glutamate had a variety of effects on local flexibility, whereas norvaline significantly decreased conformational fluctuations that allow protease cleavage. Dynamic light scattering suggests that norvaline stabilizes all interfaces in the hexamer, whereas glutamate had little effect on trimer–trimer interactions. The substrate glutamate exhibits negative cooperativity and complex allosteric regulation but has only minor effects on global GDH stability, while promoting certain local conformational fluctuations. In contrast, the substrate norvaline does not show negative cooperativity or allow allosteric regulation. Instead, norvaline significantly stabilizes the enzyme and markedly slows or prevents local conformational fluctuations that are likely to be important for cooperative effects and to determine the overall rate of hydride transfer. This suggests that homotropic allosteric regulation by the enzymatic substrate involves changes in both global stability and local flexibility of the protein.

Using research to teach an “introduction to biological thinking”

A course design for first‐year science students is described, where the focus is on the skills necessary to do science. The course uses original research projects, designed by the students, to teach a variety of skills including reading the scientific literature, hypothesis development and testing, experimental design, data analysis and interpretation, and quantitative skills and presentation of the research in a variety of formats.Biochemistry and Molecular Biology Education Vol. 39, No. 1, pp. 10–16, 2011

A National Comparison of Biochemistry and Molecular Biology Capstone Experiences.

Recognizing the increasingly integrative nature of the molecular life sciences, the American Society for Biochemistry and Molecular Biology (ASBMB) recommends that Biochemistry and Molecular Biology (BMB) programs develop curricula based on concepts, content, topics, and expected student outcomes, rather than courses. To that end, ASBMB conducted a series of regional workshops to build a BMB Concept Inventory containing validated assessment tools, based on foundational and discipline‐specific knowledge and essential skills, for the community to use. A culminating activity, which integrates the educational experience, is often part of undergraduate molecular life science programs. These “capstone” experiences are commonly defined as an attempt to measure student ability to synthesize and integrate acquired knowledge. However, the format, implementation, and approach to outcome assessment of these experiences are quite varied across the nation. Here we report the results of a nation‐wide survey on BMB capstone experiences and discuss this in the context of published reports about capstones and the findings of the workshops driving the development of the BMB Concept Inventory. Both the survey results and the published reports reveal that, although capstone practices do vary, certain formats for the experience are used more frequently and similarities in learning objectives were identified. The use of rubrics to measure student learning is also regularly reported, but details about these assessment instruments are sparse in the literature and were not a focus of our survey. Finally, we outline commonalities in the current practice of capstones and suggest the next steps needed to elucidate best practices.

A novel mechanism of V-type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis.

Bovine glutamate dehydrogenase is potently inhibited by zinc and the major impact is on Vmax suggesting a V‐type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid norvaline, which gives no evidence of subunit interactions, zinc does not inhibit. Zinc significantly decreases the size of the pre‐steady state burst in the reaction but does not affect NADPH binding in the enzyme–NADPH–glutamate complex that governs the steady state turnover, again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicates that regions in the vicinity of the antennae regions and the trimer–trimer interface become more flexible. The structures of glutamate dehydrogenase bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover, which is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V‐type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation.

Commentary: Biochemistry and Molecular Biology Educators Launch National Network.

From the †Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, ‡Department of Biochemistry and Molecular Biology, University of Richmond, Richmond, Virginia, §Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama, |Biochemistry Department, North Carolina State University, Raleigh, North Carolina, ||Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, California, and ¶Department of Chemistry and Biochemistry, University of Delware, Newark, Delware