Recruitment of faculty members in academic departments shapes the department for decades in research and teaching arenas. A diverse department is beneficial for all students as representation of underrepresented minority groups in the professoriate can inspire a greater diversity of students to pursue higher levels of education or research-focused careers. Increased diversity benefits research directly as diverse teams have been shown to have better ideas and outcomes. In 2020, our department had lower gender diversity than expected based on the pool of qualified personnel in Canada. Therefore, we altered our hiring process, primarily by redacting applications, for recruitment into entry-level tenure-track faculty positions. This resulted in the increased hiring of women (17% to 80%) with no substantial change in hiring of racially diverse individuals (50% to 40%). Overall, combined with retirements, the percentage of women faculty in the department went from 25% to 50% and the percentage of racialized faculty went from 38% to 44%. Thus, our intervention was successful in increasing the diversity of our department within a short timeframe. Our experience could provide other departments with a template for making substantive change, even in the absence of internal expertise in the area.

Lung Surfactant Protein B (SP-B) is essential for life. It is thus striking that, to this point, no method for making the full-length protein has been published and consequently we lack detailed understanding of SP-B's basic structure-function relationships, as well as an inability to make it for clinical use. The major challenge in producing SP-B lies with its exceptionally hydrophobic nature. In this work, we present a method to produce recombinant SP-B in bacteria that can be used to make the full-length protein as well as the product focused on here, which is a construct lacking the N-terminal 7 residues, rSP-B (Δ7NTC48S-SP-B-6His). The construct is produced as a fusion to Staphylococcus nuclease A (SN) in Escherichia coli C43 cells, a strain known to promote production of toxic and membrane recombinant proteins. After cleavage from SN, rSP-B is folded on column and then exchanged into the lipid or detergent system of choice. rSP-B prepared in this way exhibits the correct secondary structure and demonstrates surface activity. The yield obtained is 0.3 mg of purified rSP-B (Δ7NTC48S-SP-B-6His) per liter of initial bacterial culture. We expect this method for producing SP-B will be valuable in enabling basic research into SP-B's mechanisms, as well as possibly facilitating the inclusion of SP-B in lung surfactant formulations to treat common and frequently fatal lung conditions and in lung surfactant-based drug delivery.

α-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, α-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. 15 N- 1 H HSQC NMR results show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates over a timescale of 48 h. 15 N-edited diffusion measurements indicate that all crowders slow down the α-synuclein's diffusivity. Interestingly, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. 15 N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.

Much of the work probing antimicrobial peptide (AMP) mechanisms has focussed on how these molecules permeabilize lipid bilayers. However, AMPs must also traverse a variety of non-lipid cell envelope components before they reach the lipid bilayer. Additionally, there is a growing list of AMPs with non-lipid targets inside the cell. It is thus useful to extend the biophysical methods that have been traditionally applied to study AMP mechanisms in liposomes to the full bacteria, where the lipids are present along with the full complexity of the rest of the bacterium. This review focusses on what can be learned about AMP mechanisms from solid-state NMR of AMP-treated intact bacteria. It also touches on flow cytometry as a complementary method for measuring permeabilization of bacterial lipid membranes in whole bacteria.

The crowded and heterogeneous intracellular milieu has a concentration of macromolecules up to 400 mg/ml. The causes of macromolecular crowding effects are excluded volume and ‘soft' nonspecific interactions, such as hydrogen bonds, electrostatic, Van der Waals, and hydrophobic interactions. Some physiological consequences of macromolecular crowding on proteins are on protein structure, protein folding, phase separation, aggregation, and protein-protein interaction. Traditionally, macromolecular crowding effects has been studied in artificial crowders like Ficoll and Dextran.

Understanding how non-lipid components of bacteria affect antimicrobial peptide (AMP)-induced membrane disruption is important for a comprehensive understanding of AMP mechanisms and informing AMP-based drug development. This study investigates how lipopolysaccharide (LPS) affects membrane disruption by the AMP MSI-78 and compares the results to the effect of TP2, a cell-penetrating peptide that crosses membrane bilayers without permeabilizing them. We destabilize the LPS layer of Escherichia coli (E. coli) cells via chelation of the stabilizing divalent cations. 2H NMR spectra of E. coli demonstrate that EDTA concentrations of 2.5 mM and 9.0 mM alone have very minor effects on lipid acyl chain order. Interestingly, we find that E. coli pre-treated with 9.0 mM EDTA before treatment with MSI-78 are more sensitive to AMP-induced acyl chain disruption, indicating that intact LPS reduces MSI-78-induced membrane disruption in E. coli. Surprisingly, we also found that at the level of 2H_NMR, the peptide-induced acyl chain disruption is similar for MSI-78 and TP2, although MSI-78 permeabilizes the bilayer and TP2 does not. Furthermore, LPS disruption appears to protect the bacteria from TP2, although it sensitizes them to MSI-78.

Antimicrobial peptides (AMPs) offer advantages over conventional antibiotics; for example, bacteria develop more resistance to small-molecule antibiotics than to AMPs. The interaction of the AMPs with the lipopolysaccharide (LPS) layer of the Gram-negative bacteria cell envelope is not well understood. A MARTINI model was constructed of a Gram-negative bacterial outer membrane interacting with the AMP Magainin 2. In a 20 μs molecular dynamics (MD) simulation, the AMP diffused to the LPS layer of the cell envelope and remained there, suggesting interactions between the Magainin 2 and the LPS layer, causing the AMP to concentrate at that position. The free energy profile for the insertion of the Magainin 2 into the membrane was also calculated using umbrella sampling, which showed that the AMP positioned such that the cationic side chains of the AMP coordinated to the negatively charged phosphate groups of the LPS layer. These simulations indicate that the AMP Magainin 2 partition into the LPS layer of a bacterial membrane.

While peptides can be excellent therapeutics for several conditions, their limited in vivo half-lives have been a major bottleneck in the development of therapeutic peptides. Conjugating the peptide to an inert chemical moiety is a strategy that has repeatedly proven to be successful in extending the half-life of some therapeutics. This systematic review and meta-analysis was conducted to examine the available literature and assess it in an unbiased manner to determine which conjugates, both biological and synthetic, provide the greatest increase in therapeutic peptide half-life. Systematic searches run on PubMed, Scopus and SciFinder databases resulted in 845 studies pertaining to the topic, 16 of these were included in this review after assessment against pre-specified inclusion criteria registered on PROSPERO (#CRD42020222579). The most common reasons for exclusion were non-IV administration and large peptide size. Of the 16 studies that were included, a diverse suite of conjugates that increased half-life from 0.1 h to 33.57 h was identified. Amongst these peptides, the largest increase in half-life was seen when conjugated with glycosaminoglycans. A meta-analysis of studies that contained fatty acid conjugates indicated that acylation contributed to a statistically significant extension of half-life. Additionally, another meta-analysis followed by a sensitivity analysis suggested that conjugation with specifically engineered recombinant peptides might contribute to a more efficient extension of peptide half-life as compared to PEGylation. Moreover, we confirmed that while polyethylene glycol is a good synthetic conjugate, its chain length likely has an impact on its effectiveness in extending half-life. Furthermore, we found that most animal studies do not include as much detail when reporting findings as compared to human studies. Inclusion of additional experimental detail on aspects such as independent assessment and randomization may be an easily accomplished strategy to drive more conjugated peptides towards clinical studies.

Solid state NMR has been tremendously useful in characterizing the structure and dynamics of model membranes composed of simple lipid mixtures. Model lipid studies employing solid state NMR have included important work revealing how membrane bilayer structure and dynamics are affected by molecules such as antimicrobial peptides (AMPs). However, solid state NMR need not be applied only to model membranes, but can also be used with living, intact cells. NMR of whole cells holds promise for helping resolve some unsolved mysteries about how bacteria interact with AMPs. This mini-review will focus on recent studies using 2H NMR to study how treatment with AMPs affect membranes in intact bacteria.

The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (Rg ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.

Otosclerosis is a bone disorder of the otic capsule and common form of late-onset hearing impairment. Considered a complex disease, little is known about its pathogenesis. Over the past 20 years, ten autosomal dominant loci (OTSC1-10) have been mapped but no genes identified. Herein, we map a new OTSC locus to a 9.96 Mb region within the FOX gene cluster on 16q24.1 and identify a 15 bp coding deletion in Forkhead Box L1 co-segregating with otosclerosis in a Caucasian family. Pre-operative phenotype ranges from moderate to severe hearing loss to profound sensorineural loss requiring a cochlear implant. Mutant FOXL1 is both transcribed and translated and correctly locates to the cell nucleus. However, the deletion of 5 residues in the C-terminus of mutant FOXL1 causes a complete loss of transcriptional activity due to loss of secondary (alpha helix) structure. FOXL1 (rs764026385) was identified in a second unrelated case on a shared background. We conclude that FOXL1 (rs764026385) is pathogenic and causes autosomal dominant otosclerosis and propose a key inhibitory role for wildtype Foxl1 in bone remodelling in the otic capsule. New insights into the molecular pathology of otosclerosis from this study provide molecular targets for non-invasive therapeutic interventions.

The histidine-rich antimicrobial peptides (AMPs) Gad-1 and Gad-2, from paralogous genes in cod, provide an opportunity to examine the effect of charge and non-electrostatic factors on peptide-vesicle interaction and on peptide antimicrobial activity. In this study, the dependence of vesicle ζ-potential on peptide concentration has been used to examine the binding of these peptides to model vesicle surfaces at pH=5.0, for which the charges of Gad-1 and Gad-2 are +8 and +5 respectively, and at pH=7.0 where their charges are +3 and +1 respectively. Interpreting the observed ζ-potential behaviors as examples of Langmuir adsorption isotherms, it is possible to infer the equilibrium constant for peptide-vesicle binding, the fraction of peptide bound at low peptide concentration, and the maximum peptide-to-lipid ratio when the vesicle surface is saturated at high peptide concentration. For both peptides, higher peptide charge is found to be correlated with a lower fraction of peptide being bound to vesicle surfaces at low peptide concentration and with a smaller maximum bound-peptide-to-lipid ratio at high peptide concentration. The equilibrium binding constant, on the other hand, is more strongly correlated with peptide sequence than with charge. Gad-1, which has been shown to be more biologically active than Gad-2, displayed a significantly higher equilibrium binding constant. These observations suggest that while maximum peptide density on the vesicle surface is limited by electrostatic interactions, the free energy of peptide binding, like the observed antimicrobial activities of the Gad peptides, is also sensitive to other peptide factors which might, for example, influence hydrophobic interactions.

Gad-1 and Gad-2 are helical, histidine-rich antimicrobial peptides (AMPs) from paralogous genes in cod. N-15 and H-2 solid state nuclear magnetic resonance (NMR) were used to characterize their lipid-bound structures and lipid interactions. Gad-1 was found to position in-plane in POPC: POPG bilayers. Gad-1 displayed greater effects than Gad-2 on lipid acyl chain order of POPE: POPG and POPE: POPG: CL bilayers, in keeping with its greater activity against E. coli. The effect of Gad-1 and Gad-2 on lipid bilayer order was only weakly affected by changes in pH, and hence changes in histidine charge. This was somewhat surprising for Gad-2 as this peptide's biological activity has been shown to be greater at low pH and thus the finding may point to the existence of functional interactions with non-lipid components of bacteria. The incorporation of cardiolipin into POPE: POPG bilayers in such a way as to preserve the overall charge of the bilayers did not alter Gad-1's effects on lipid acyl chain order parameters, which report on motions on the 10(-5) s timescale. When cardiolipin and Gad-1 were both present, there were subtle changes on membrane dynamics at other timescales.

Protein interactions take place in the crowded and heterogeneous intracellular milieu with macromolecular concentrations up to 400 mg/ml. The role of excluded-volume effects and soft non-specific interactions in macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran. Our measurements of flow properties show, however, that artificial crowders such as Ficoll have starkly different rheological characteristics from a complex biological crowder, bacterial cell lysate, at comparable concentrations. Moreover, biological crowders are a heterogeneous collection of biomolecules with a distribution of sizes, shapes and charge; these differences likely also affect translational diffusion of biomolecules within the cell. In this study, we examine the effects of size distribution and charge of a complex biological crowder on the diffusion of polymers and ask if this is similar to what is seen for artificial crowders like Ficoll. To answer this question, we use a top-down experiment using bacterial cell lysate as the crowder. To test the dynamical properties of polymer in the lysate environment, we measure the translational diffusion of the test polymer polyethylene glycol (PEG) using diffusion NMR. While PEG is not a biomolecule, it is a useful analog for a disordered protein. Our rheological tests show that while Ficoll is nearly Newtonian even at high concentrations, bacterial cell lysate is markedly viscoelastic. Results will provide a better understanding of the effects of complex biological crowders on polymer diffusion.

The lipid bilayer disrupting effect of antimicrobial peptides (AMPs) has been widely studied in model-lipid systems by applying biophysical techniques such as 2H NMR spectroscopy. Real bacteria cell envelopes contain non-lipid components, such as peptidoglycan, and thus it is important to assess the effects of such non-lipid components on the lipid-disrupting effects of AMPs. To this end, our group and other have developed methods that promote uptake of deuterium-labeled acyl chains in bacterial cells to produce 2H-membrane-enriched Bacillus subtilis. In this work, we studied changes in the static 2H NMR spectra of B. subtilis induced by the AMPs MSI-78 and BP100. Addition of both AMPs resulted in the increase of lipid acyl chain disorder consistent with disruption of the bacterial membrane. In addition, the peptide to lipid molar ratios (P:L) that give rise to observable effects fall between the P:L molar ratios necessary to generate membrane disruption in model-lipid-only systems and the P:L molar ratios needed to inhibit bacterial cell growth. This observation supports a role for the non-lipid components in modulating the AMP-lipid interactions.

Antimicrobial Peptides (AMPs) have been studied for more than two decades because of their promise to help overcome the problem of resistance to conventional antibiotics. However, AMPs have not been as successful as hoped, likely because we lack a detailed understanding of their mechanisms of action. Many biophysical studies of AMPs are performed in model membrane systems, composed of just one or two lipid components. Such studies have shown that membrane permeabilization is a common mechanism of action. Yet we don’t fully understand their connection between the behavior of AMPs in model lipids, where membrane permeabilization is seen and the behavior in real bacteria, where AMPs are observed to inhibit cell growth. Bacteria have an extra non-lipid component in their cell envelopes. It is thus possible that interactions between AMPs and non-lipid components of the cell envelope are important to their mechanisms of action. Specifically, the focus of this study is to find out if the lipopolysaccharide (LPS) outer membrane layer of Gram-negative bacteria promotes or inhibits AMP-induced membrane disruption. This work employed the magainin analog, MSI-78. We disrupt the lipopolysaccharide layer of Gram-negative bacteria (E.coli) via chelation of the stabilizing divalent cations. Then, we use deuterium NMR of deuterated intact bacteria to observe how AMP-induced acyl chain disruption is affected by LPS layer destabilization. In addition to 2H NMR, we do light microscopy and flow cytometry to confirm the EDTA disruption of LPS.

We live in a time when there is an astounding array of techniques to unveil the inner workings of the molecules found in and around our cells. Many of these techniques were originally developed for probing systems with simple compositions of one or two types of biomolecule. However, there is a growing effort to extend such techniques to the much more complex milieu in which the molecules of interest normally function in living organisms. Nuclear magnetic resonance (NMR) is one powerful technique for probing the detailed structure and dynamics of biomolecules including proteins and lipids. While traditionally applied to samples of simple composition, NMR is increasingly being applied in more complex contexts including to intact, whole cells. Deuterium (H-2)-NMR has been of great utility in probing the structure and dynamics of H-2-labelled lipids in bilayers; and it has also been successfully applied to whole cells. This chapter will introduce readers to this approach as applied to bacteria, as well as highlight some of the key findings from recent years, in particular with respect to how antimicrobial peptides (AMPs) interact with membranes in bacteria.

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B’s structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B’s structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B’s “central loop” and “hinge” regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B’s essential functions.