The Art of Science Communication-A Novel Approach to Science Communication Training.

Effective communication is a requisite skill for scientists. However, formalized training in this area is often unavailable for members of the scientific community. As one approach to combat this problem, the American Society for Biochemistry and Molecular Biology (ASBMB) developed The Art of Science Communication, an eight-week-long online course that provides facilitated instruction on how to communicate science in an oral format. The course is offered three times a year, and as of December 2017, nearly 200 individuals from all career stages have taken part in it. The course completion rate is currently 60%, a rate three to five times as high as the average for similar Massive Open Online Courses (MOOCs). Participants have indicated that taking the course has improved their ability to communicate about their research, and that the skills and lessons learned have benefited them professionally. Moving forward, we are examining approaches that will help us improve the course and expand its reach throughout the scientific community. This article details the development of the course and examines the role and potential of such training within the larger scientific community.

Analysis of the bacterial luciferase mobile loop by replica-exchange molecular dynamics.

Bacterial luciferase contains an extended 29-residue mobile loop. Movements of this loop are governed by binding of either flavin mononucleotide (FMNH2) or polyvalent anions. To understand this process, loop dynamics were investigated using replica-exchange molecular dynamics that yielded conformational ensembles in either the presence or absence of FMNH2. The resulting data were analyzed using clustering and network analysis. We observed the closed conformations that are visited only in the simulations with the ligand. Yet the mobile loop is intrinsically flexible, and FMNH2 binding modifies the relative populations of conformations. This model provides unique information regarding the function of a crystallographically disordered segment of the loop near the binding site. Structures at or near the fringe of this network were compatible with flavin binding or release. Finally, we demonstrate that the crystallographically observed conformation of the mobile loop bound to oxidized flavin was influenced by crystal packing. Thus, our study has revealed what we believe are novel conformations of the mobile loop and additional context for experimentally determined structures.

Two lysine residues in the bacterial luciferase mobile loop stabilize reaction intermediates.

Bacterial luciferase catalyzes the reaction of FMNH(2), O(2), and a long chain aliphatic aldehyde, yielding FMN, carboxylic acid, and blue-green light. The most conserved contiguous region of the primary sequence corresponds to a crystallographically disordered loop adjacent to the active center (Fisher, A. J., Raushel, F. M., Baldwin, T. O., and Rayment, I. (1995) Biochemistry 34, 6581-6586; Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., and Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968). Deletion of the mobile loop does not alter the chemistry of the reaction but decreases the total quantum yield of bioluminescence by 2 orders of magnitude (Sparks, J. M., and Baldwin, T. O. (2001) Biochemistry 40, 15436-15443). In this study, we attempt to localize the loss of activity observed in the loop deletion mutant to individual residues in the mobile loop. Using alanine mutagenesis, the effects of substitution at 15 of the 29 mobile loop residues were examined. Nine of the point mutants had reduced activity in vivo. Two mutations, K283A and K286A, resulted in a loss in quantum yield comparable with that of the loop deletion mutant. The bioluminescence emission spectrum of both mutants was normal, and both yielded the carboxylic acid chemical product at the same efficiency as the wild-type enzyme. Substitution of Lys(283) with alanine resulted in destabilization of intermediate II, whereas mutation of Lys(286) had an increase in exposure of reaction intermediates to a dynamic quencher. Based on a model of the enzyme-reduced flavin complex, the two critical lysine residues are adjacent to the quininoidal edge of the isoalloxazine.

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli.

Unlike the vast majority of flavoenzymes, bacterial luciferase requires an exogenous source of reduced flavin mononucleotide for bioluminescence activity. Within bioluminescent bacterial cells, species-specific oxidoreductases are believed to provide reduced flavin for luciferase activity. The source of reduced flavin in Escherichia coli-expressing bioluminescence is not known. There are two candidate proteins potentially involved in this process in E. coli, a homolog of the Vibrio harveyi Frp oxidoreductase, NfsA, and a luxG type oxidoreductase, Fre. Using single gene knock-out strains, we show that deletion of fre decreased light output by greater than two orders of magnitude, yet had no effect on luciferase expression in E. coli. Purified Fre is capable of supporting bioluminescence in vitro with activity comparable to that with the endogenous V. harveyi reductase (Frp), using either FMN or riboflavin as substrate. In a pull-down experiment, we found that neither Fre nor Frp co-purify with luciferase. In contrast to prior work, we find no evidence for stable complex formation between luciferase and oxidoreductase. We conclude that in E. coli, an enzyme primarily responsible for riboflavin reduction (Fre) can also be utilized to support high levels of bioluminescence.

Demonstration of two independently folding domains in the alpha subunit of bacterial luciferase by preferential ligand binding-induced stabilization.

The alpha subunit of bacterial luciferase unfolds and refolds reversibly by a three-state mechanism in urea-containing buffer. It has been proposed that the three-state unfolding of the alpha subunit arises from a stepwise unfolding of a C-terminal folding domain at lower concentrations of urea, followed by unfolding of the N-terminal domain at higher concentrations of urea (Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145). The location of an anion binding site in the proposed N-terminal folding domain allowed the folding mechanism to be probed in the context of the intact polypeptide. Anions preferentially stabilized the N-terminal domain in a concentration-dependent manner. The polyvalent anions sulfate and phosphate were found to be more stabilizing than monovalent chloride ion. Cations did not show a similar stabilizing effect, demonstrating that the stabilization was due to the anions alone. The purified N-terminal domain prepared by limited proteolysis and anion exchange chromatography was found to refold cooperatively with a midpoint approximately that of the second unfolding transition of the alpha subunit. Phosphate ion stabilized this fragment to roughly the same extent as it did the alpha subunit. The results presented are consistent with the proposed two-domain folding model and demonstrate that anion binding to the N-terminal folding domain stabilizes the alpha subunit of bacterial luciferase.

Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme which catalyzes a light-producing reaction in Vibrio harveyi. In addition to the alphabeta enzyme, the beta subunit can self-associate to form a stable but inactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]. The studies reported here were undertaken to explore the role of the subunit interface in the conformational stability of the enzyme. To this end, we constructed four mutant heterodimers in which residues at the subunit interface were changed in an effort to alter the volume of an apparent solvent accessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for the heterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779]. However, we found that unfolding for the wild-type and mutant luciferases is better described by a four-state model. This change in the proposed mechanism of unfolding is based on observation of residual structure in the subunits following dissociation of the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly, no change in the DeltaG2H2O value for subunit dissociation and no measurable change in the equilibrium dissociation constant relative to that of the wild-type heterodimer. However, the DeltaG1H2O value for the formation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants, indicating that mutations at the interface can alter the stability of a region of the alpha subunit that is distant from the interface. We conclude that the interface region communicates with the distal domains of this subunit, probably through the active center region of the enzyme.