EMMAs: Implementation and Assessment of a Suite of Cross-Disciplinary, Case-Based High School Activities to Explore Three-Dimensional Molecular Structure, Noncovalent Interactions, and Molecular Dynamics.

Students frequently develop misconceptions about noncovalent interactions that make it challenging for them to appropriately interpret aspects of molecular structure and interactions critical to myriad applications. We hypothesized that computational molecular modeling and visualization could provide a valuable approach to help address these core misconceptions when students are first exposed to these concepts in secondary school chemistry courses. Here, we present a series of activities exploring biomolecular drug-target interactions using molecular visualization software and an introduction to molecular dynamics methods that were implemented in secondary school classrooms. A pre- and postsurvey approach that incorporated Likert response type, written free response, and drawing-based items demonstrated that students gained an enhanced conceptualization of intermolecular interactions, particularly related to aspects of shape complementarity, after completing the activities. Students also expressed increased comfort with and facility in utilizing different three-dimensional representations of molecules in their postsurvey responses. The activities led to an increased appreciation of interdisciplinary connections of chemistry with mathematics and physics. Overall, the modular activities presented provide a relatively time-efficient and accessible manner to help promote an understanding of a traditionally challenging topic for beginning chemistry students while introducing them to contemporary research tools.

Qualitative and Quantitative Changes to Escherichia coli during Treatment with Magainin 2 Observed in Native Conditions by Atomic Force Microscopy.

The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides.

Studies attempting to characterize the membrane translocation of antimicrobial and cell-penetrating peptides are frequently limited by the resolution of conventional light microscopy. This study shows that spheroplasts provide a valuable approach to overcome these limits. Spheroplasts produce less ambiguous images and allow for more systematic analyses of localization. Data collected with spheroplasts are consistent with studies using normal bacterial cells and imply that a particular peptide may not always follow the same mechanism of action.

Modular analysis of hipposin, a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments.

Antimicrobial peptides continue to garner attention as potential alternatives to conventional antibiotics. Hipposin is a histone-derived antimicrobial peptide (HDAP) previously isolated from Atlantic halibut. Though potent against bacteria, its antibacterial mechanism had not been characterized. The mechanism of this peptide is particularly interesting to consider since the full hipposin sequence contains the sequences of parasin and buforin II (BF2), two other known antimicrobial peptides that act via different antibacterial mechanisms. While parasin kills bacteria by inducing membrane permeabilization, buforin II enters cells without causing significant membrane disruption, harming bacteria through interactions with intracellular nucleic acids. In this study, we used a modular approach to characterize hipposin and determine the role of the parasin and buforin II fragments in the overall hipposin mechanism. Our results show that hipposin kills bacteria by inducing membrane permeabilization, and this membrane permeabilization is promoted by the presence of the N-terminal domain. Portions of hipposin lacking the N-terminal sequence do not cause membrane permeabilization and function more similarly to buforin II. We also determined that the C-terminal portion of hipposin, HipC, is a cell-penetrating peptide that readily enters bacterial cells but has no measurable antimicrobial activity. HipC is the first membrane active histone fragment identified that does not kill bacterial or eukaryotic cells. Together, these results characterize hipposin and provide a useful starting point for considering the activity of chimeric peptides made by combining peptides with different antimicrobial mechanisms. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Heteromultimerization of prokaryotic bacterial cyclic nucleotide-gated (bCNG) ion channels, members of the mechanosensitive channel of small conductance (MscS) superfamily.

Traditionally, prokaryotic channels are thought to exist as homomultimeric assemblies, while many eukaryotic ion channels form complex heteromultimers. Here we demonstrate that bacterial cyclic nucleotide-gated channels likely form heteromultimers in vivo. Heteromultimer formation is indicated through channel modeling, pull-down assays, and real-time polymerase chain reaction analysis. Our observations demonstrate that prokaryotic ion channels can display complex behavior and regulation akin to that of their eukaryotic counterparts.

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms.

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.

Electrostatics-Based Computational Design and Experimental Analysis of Buforin II Antimicrobial Peptide Variants with Increased DNA Affinities.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics in the treatment of bacterial infections in part due to their targeting of generic bacterial structures that make it more difficult to develop drug resistance. In this study, we introduce and implement a design workflow to develop more potent AMPs by improving their electrostatic interactions with DNA, which is a putative intracellular target. Using the existing membrane-translocating AMP buforin II (BF2) as a starting point, we use a computational workflow that integrates electrostatic charge optimization, continuum electrostatics, and molecular dynamics simulations to suggest peptide positions at which a neutral BF2 residue could be substituted with arginine to increase DNA-binding affinity either significantly or minimally, with the latter choice done to determine whether AMP binding affinity depends on charge distribution and not just overall monopole. Our analyses predicted that T1R and L8R BF2 variants would yield substantial and minimal increases in DNA-binding affinity, respectively. These predictions were validated with experimental peptide-DNA binding assays with additional computational analyses providing structural insights. Additionally, experimental measurements of antimicrobial potency showed that a design to increase DNA binding can also yield greater potency. As a whole, this study takes initial steps to support the idea that (i) a design strategy aimed to increase AMP binding affinity to DNA by focusing only on electrostatic interactions can improve AMP potency and (ii) the effect on DNA binding of increasing the overall peptide monopole via arginine substitution depends on the position of the substitution. More broadly, this design strategy is a novel way to increase the potency of other membrane-translocating AMPs that target nucleic acids.

Mechanosensitive behavior of bacterial cyclic nucleotide gated (bCNG) ion channels: Insights into the mechanism of channel gating in the mechanosensitive channel of small conductance superfamily.

We have recently identified and characterized the bacterial cyclic nucleotide gated (bCNG) subfamily of the larger mechanosensitive channel of small conductance (MscS) superfamily of ion channels. The channel domain of bCNG channels exhibits significant sequence homology to the mechanosensitive subfamily of MscS in the regions that have previously been used as a hallmark for channels that gate in response to mechanical stress. However, we have previously demonstrated that three of these channels are unable to rescue Escherichiacoli from osmotic downshock. Here, we examine an additional nine bCNG homologues and further demonstrate that the full-length bCNG channels are unable to rescue E. coli from hypoosmotic stress. However, limited mechanosensation is restored upon removal of the cyclic nucleotide binding domain. This indicates that the C-terminal domain of the MscS superfamily can drive channel gating and further highlight the ability of a superfamily of ion channels to be gated by multiple stimuli.

Ss-bCNGa: a unique member of the bacterial cyclic nucleotide gated (bCNG) channel family that gates in response to mechanical tension.

Bacterial cyclic nucleotide gated (bCNG) channels are generally a nonmechanosensitive subset of the mechanosensitive channel of small conductance (MscS) superfamily. bCNG channels are composed of an MscS channel domain, a linking domain, and a cyclic nucleotide binding domain. Among bCNG channels, the channel domain of Ss-bCNGa, a bCNG channel from Synechocystis sp. PCC 6803, is most identical to Escherichia coli (Ec) MscS. This channel also exhibits limited mechanosensation in response to osmotic downshock assays, making it the only known full-length bCNG channel to respond to hypoosmotic stress. Here, we compare and contrast the ability of Ss-bCNGa to gate in response to mechanical tension with Se-bCNG, a nonmechanosensitive bCNG channel, and Ec-MscS, a prototypical mechanosensitive channel. Compared with Ec-MscS, Ss-bCNGa only exhibits limited mechanosensation, which is most likely a result of the inability of Ss-bCNGa to form the strong lipid contacts needed for significant function. Unlike Ec-MscS, Ss-bCNGa displays a mechanical response that increases with protein expression level, which may result from channel clustering driven by interchannel cation-π interactions.

Defining the role of the tension sensor in the mechanosensitive channel of small conductance.

Mutations that alter the phenotypic behavior of the Escherichia coli mechanosensitive channel of small conductance (MscS) have been identified; however, most of these residues play critical roles in the transition between the closed and open states of the channel and are not directly involved in lipid interactions that transduce the tension response. In this study, we use molecular dynamic simulations to predict critical lipid interacting residues in the closed state of MscS. The physiological role of these residues was then investigated by performing osmotic downshock assays on MscS mutants where the lipid interacting residues were mutated to alanine. These experiments identified seven residues in the first and second transmembrane helices as lipid-sensing residues. The majority of these residues are hydrophobic amino acids located near the extracellular interface of the membrane. All of these residues interact strongly with the lipid bilayer in the closed state of MscS, but do not face the bilayer directly in structures associated with the open and desensitized states of the channel. Thus, the position of these residues relative to the lipid membrane appears related to the ability of the channel to sense tension in its different physiological states.

Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels.

Studies of bacterial ion channels have provided significant insights into the structure-function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.

A fully integrated undergraduate introductory biology and chemistry course with a community-based focus II: Assessment of course effectiveness.

In the Fall of 2016, we created an integrated introductory biology/chemistry course for first-semester students at Wellesley College. This course was designed to meaningfully integrate chemistry and molecular cell biology while also building community and fostering mentorship both inside and outside the classroom. Here, we describe the assessment of this integrated course through a combination of multivariate analyses of student transcript data and student focus group discussions. Our assessment found that the integrated course provided a strongly collaborative working environment for students that provided them with skills that promoted success in future courses. Along with a rigorous consideration of the interplay between biology and chemistry, these skills appeared to support positive longer-term student outcomes. In particular, we observed significant impacts on student persistence into and performance in intermediate and advanced courses. Students from the integrated course were also significantly more likely to declare a major in biochemistry than students who took one of the traditional introductory courses. In addition, our assessment also noted the importance of a cohesive instructional team and broad faculty participation in the success and sustainability of the course.

A fully integrated undergraduate introductory biology and chemistry course with a community-based focus I: Vision, design, implementation, and development.

We describe a first-semester, integrated, introductory biology and chemistry course for undergraduates at Wellesley College in Wellesley, MA, USA. Our vision was to create a supportive learning community in which students could comfortably make connections between scientific disciplines as they learned necessary content for subsequent courses, further developed problem solving, communication, and laboratory skills, and meaningfully connected with other students and with faculty during their first semester in college. Through highlighting five guiding principles that are central to the course, we describe the integrated course structure and content as well as our efforts to build community, provide support, and engage students in building skills crucial to scientists. We also highlight features of this course and institutional policies that facilitated its logistical and collaborative implementation that can be adapted to fit the needs, goals, and constraints of a diverse range of institutions. A companion article describes an assessment of our course in achieving academic and community building goals.

Computationally Modeling Electrostatic Binding Energetics in a Crowded, Dynamic Environment: Physical Insights from a Peptide-DNA System.

The cell is a crowded place, and it may be crucial at times to account for the local environment when studying determinants of molecular recognition. In this work, we use continuum electrostatics calculations on snapshots extracted from molecular dynamics simulations to understand how various aspects of a crowded environment affect electrostatic binding energies between the antimicrobial peptide buforin II and DNA. By comparing multiple models for representing crowding, sequentially introducing layers of model complexity for maximum control, we explore how electrostatic binding energetics depend on crowder physical properties, the sampling of the binding partners and crowder molecules, and the treatment of bulk solvent. We show that physical characteristics can combine to create an interplay of competing effects in this highly charged system. For example, increased ionic strength screening due to crowding partially cancels out the reduced solvent screening due to water depletion. We also quantify the effect of crowders' charge distributions on binding energetics. While we focus on electrostatic effects of crowding on binding, we begin to consider nonpolar components as well, and we implement a thermodynamic cycle accounting for both bound and unbound states to show the necessity of adequate crowder sampling in future studies. The insights developed here provide a rich starting point for experiments to further explore these competing effects and, ultimately, to rationally modulate molecular recognition in the complex cellular environment.

Using fluorescence microscopy to shed light on the mechanisms of antimicrobial peptides.

Antimicrobial peptides (AMPs) are promising in the fight against increasing bacterial resistance, but the development of AMPs with enhanced activity requires a thorough understanding of their mechanisms of action. Fluorescence microscopy is one of the most flexible and effective tools to characterize AMPs, particularly in its ability to measure the membrane interactions and cellular localization of peptides. Recent advances have increased the scope of research questions that can be addressed via microscopy through improving spatial and temporal resolution. Unique combinations of fluorescent labels and dyes can simultaneously consider different aspects of peptide-membrane interaction mechanisms. This review emphasizes the central role that fluorescence microscopy will continue to play in the interrogation of AMP structure-function relationships and the engineering of more potent peptides.

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization.

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.

Confirming the revised C-terminal domain of the MscL crystal structure.

The structure of the C-terminal domain of the mechanosensitive channel of large conductance (MscL) has generated significant controversy. As a result, several structures have been proposed for this region: the original crystal structure (1MSL) of the Mycobacterium tuberculosis homolog (Tb), a model of the Escherichia coli homolog, and, most recently, a revised crystal structure of Tb-MscL (2OAR). To understand which of these structures represents a physiological conformation, we measured the impact of mutations to the C-terminal domain on the thermal stability of Tb-MscL using circular dichroism and performed molecular dynamics simulations of the original and the revised crystal structures of Tb-MscL. Our results imply that this region is helical and adopts an alpha-helical bundle conformation similar to that observed in the E. coli MscL model and the revised Tb-MscL crystal structure.

Effect of lipid composition on buforin II structure and membrane entry.

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.

Investigating the nucleic acid interactions and antimicrobial mechanism of buforin II.

Buforin II (BF2) is an antimicrobial peptide that is hypothesized to kill bacteria by entering cells and binding nucleic acids. To further investigate this proposed mechanism, we used computer modeling and experimental measurements to consider the interactions between BF2 and DNA. Computational and experimental results imply that the peptide forms specific interactions with DNA. Moreover, we observe a general correlation between DNA affinity and antimicrobial activity for a series of BF2 variants. Thus, our results support the proposed mechanism for BF2 and provide a useful approach for evaluating the nucleic acid interactions of other antimicrobial peptides.

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.