Phage Coat Protein Research Articles

Engineered M13-Derived Bacteriophages Capable of Gold Nanoparticle Synthesis and Nanogold Manipulations

For years, gold nanoparticles (AuNPs) have been widely used in medicine and industry. Although various experimental procedures have been reported for their preparation and manipulation, none of them is optimal for all purposes. In this work, we engineered the N-terminus of the pIII minor coat protein of bacteriophage (phage) M13 to expose a novel HLYLNTASTHLG peptide that effectively and specifically binds gold. In addition to binding gold, this engineered phage could synthesize spherical AuNPs of 20 nm and other sizes depending on the reaction conditions, aggregate them, and precipitate gold from a colloid, as revealed by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as ultraviolet–visible (UV–vis) and Fourier-transform infrared (FTIR) spectroscopic methods. We demonstrated that the engineered phage exposing a foreign peptide selected from a phage-displayed library may serve as a sustainable molecular factory for both the synthesis of the peptide and the subsequent overnight preparation of AuNPs from gold ions at room temperature and neutral pH in the absence of strong reducing agents, such as commonly used NaBH4. Taken together, the results suggest the potential applicability of the engineered phage and the new, in vitro-identified gold-binding peptide in diverse biomimetic manipulations.

Proximity-driven site-specific cyclization of phage-displayed peptides

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

Implication of polymerase recycling for nascent transcript quantification by live cell imaging.

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state.

Harnessing filamentous phages for enhanced stroke recovery.

Stroke poses a critical global health challenge, leading to substantial morbidity and mortality. Existing treatments often miss vital timeframes and encounter limitations due to adverse effects, prompting the pursuit of innovative approaches to restore compromised brain function. This review explores the potential of filamentous phages in enhancing stroke recovery. Initially antimicrobial-centric, bacteriophage therapy has evolved into a regenerative solution. We explore the diverse role of filamentous phages in post-stroke neurological restoration, emphasizing their ability to integrate peptides into phage coat proteins, thereby facilitating recovery. Experimental evidence supports their efficacy in alleviating post-stroke complications, immune modulation, and tissue regeneration. However, rigorous clinical validation is essential to address challenges like dosing and administration routes. Additionally, genetic modification enhances their potential as injectable biomaterials for complex brain tissue issues. This review emphasizes innovative strategies and the capacity of filamentous phages to contribute to enhanced stroke recovery, as opposed to serving as standalone treatment, particularly in addressing stroke-induced brain tissue damage.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

A nanoparticle vaccine displaying the ookinete PSOP25 antigen elicits transmission-blocking antibody response against Plasmodium berghei

BackgroundSafe and effective vaccines are crucial for the control and eventual elimination of malaria. Novel approaches to optimize and improve vaccine efficacy are urgently required. Nanoparticle-based delivery platforms are considered potent and powerful tools for vaccine development.MethodsIn this study, we developed a transmission-blocking vaccine against malaria by conjugating the ookinete surface antigen PSOP25 to the Acinetobacter phage coat protein AP205, forming virus-like particles (VLPs) using the SpyTag/SpyCatcher adaptor system. The combination of AP205-2*SpyTag with PSOP25-SpyCatcher resulted in the formation of AP205-PSOP25 complexes (VLP-PSOP25). The antibody titers and avidity of serum from each immunization group were assessed by ELISA. Western blot and IFA were performed to confirm the specific reactivity of the elicit antisera to the native PSOP25 in Plasmodium berghei ookinetes. Both in vitro and in vivo assays were conducted to evaluate the transmission-blocking activity of VLP-PSOP25 vaccine.ResultsImmunization of mice with VLP-PSOP25 could induced higher levels of high-affinity antibodies than the recombinant PSOP25 (rPSOP25) alone or mixtures of untagged AP205 and rPSOP25 but was comparable to rPSOP25 formulated with alum. Additionally, the VLP-PSOP25 vaccine enhanced Th1-type immune response with remarkably increased levels of IgG2a subclass. The antiserum generated by VLP-PSOP25 specifically recognizes the native PSOP25 antigen in P. berghei ookinetes. Importantly, antisera generated by inoculation with the VLP-PSOP25 could inhibit ookinete development in vitro and reduce the prevalence of infected mosquitoes or oocyst intensity in direct mosquito feeding assays.ConclusionsAntisera elicited by immunization with the VLP-PSOP25 vaccine confer moderate transmission-reducing activity and transmission-blocking activity. Our results support the utilization of the AP205-SpyTag/SpyCatcher platform for next-generation TBVs development.Graphical abstract

Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli.

An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.

The Use of Phage Antibodies for Microbial Cells Detection (Review)

Phage antibody display technology has revolutionized the field of bacterial immunodetection. This technology allows the expression of an antibody fused to the coat protein of a filamentous bacteriophage. The use of phage display makes it possible to obtain high-affinity antibodies by passing the stage of animal immunization, reducing the time for obtaining stable antibody-producing clones from several months to several weeks, significantly reducing the cost of the process. These advantages make phage antibodies an important tool for bacterial detection. The paper presents a brief description of the technological methods for obtaining phage antibodies to microbial cells. The possibilities and prospects for using phage antibodies as a selective agent in analytical systems, including biosensors, are discussed.

Temperature-specific adaptations and genetic requirements in a biofilm formed by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen often associated with nosocomial infections that are made more severe by this bacterium's ability to form robust biofilms. A biofilm is a microbial community encompassing cells embedded within an extracellular polymeric substrate (EPS) matrix that is typically secreted by the encased microbial cells. Biofilm formation is influenced by several environmental cues, and temperature fluctuations are likely to be an important stimulus in the lifecycle of P. aeruginosa as it transitions between life in aquatic or soil environments to sites of infection in the human host. Previous work has demonstrated that human body temperature can induce a shift in the biofilm EPS relative to room temperature growth, resulting in an incorporation of a filamentous phage coat protein into the biofilm EPS. In this study, we sought to identify adaptations enabling biofilm formation at room temperature or temperatures mimicking the natural environment of P. aeruginosa (23°C and 30°C) relative to temperatures mimicking life in the human host (37°C and 40°C). We identified higher biofilm: biomass ratios at lower temperatures on certain substrates, which correlated with a higher relative abundance of apparent polysaccharide EPS content. However, the known genes for EPS polysaccharide production in P. aeruginosa PA14 did not appear to be specifically important for temperature-dependent biofilm adaptation, with the pelB gene appearing to be generally important and the algD gene being generally expendable in all conditions tested. Instead, we were able to identify two previously uncharacterized hypothetical proteins (PA14_50070 and PA14_67550) specifically required for biofilm formation at 23°C and/or 30°C relative to temperatures associated with the human host. These unstudied contributors to biofilm integrity may have been previously overlooked since most P. aeruginosa biofilm studies tend to use 37°C growth temperatures. Overall, our study demonstrates that temperature shifts can have dramatic impacts on biofilm structure and highlights the importance of studying environment-specific adaptations in biofilm physiology.

Improved extracellular vesicle-based mRNA delivery for familial hypercholesterolemia treatment.

Extracellular vesicle (EV)-based low-density lipoprotein receptor (Ldlr) mRNA delivery showed excellent therapeutic effects in treating familial hypercholesterolemia (FH). Nevertheless, the loading inefficiency of EV-based mRNA delivery presents a significant challenge. Recently, RNA-binding proteins (RBPs) have been fused to EV membrane proteins for selectively encapsulating targeted RNAs to promote loading efficiency. However, the strong interaction between therapeutic RNAs and RBPs prevents RNA release from endosomes to the cytosol in the recipient cells. In this study, an improved strategy was developed for efficient encapsulation of Ldlr mRNA into EVs in donor cells and controllable release in recipient cells. Methods: The MS2 bacteriophage coat protein (CD9-MCP) fusion protein, Ldlr mRNA, and a customized MS2 containing RNA aptamer base-pair matched with Ldlr mRNA were expressed in donor cells. Cells receiving the above therapeutic EVs were simultaneously treated with EVs containing "Ldlr releaser" with a sequence similar to the recognition sites in Ldlr mRNA. Therapeutic effects were analyzed in Ldlr-/- mice receiving EV treatments via the tail vein. Results: In vitro experiments demonstrated improved loading efficiency of Ldlr mRNA in EVs via MS2-MCP interaction. Treatment of "Ldlr releaser" competitively interacted with MS2 aptamer with higher affinity and released Ldlr mRNA from CD9-MCP for efficient translation. When the combinatory EVs were delivered into recipient hepatocytes, the robust LDLR expression afforded therapeutic benefits in Ldlr-/- mice. Conclusion: We proposed an EV-based mRNA delivery strategy for enhanced encapsulation of therapeutic mRNAs in EVs and RNA release into the cytosol for translation in recipient cells with great potential for gene therapy.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

The use of phage display systems to combat infectious diseases in poultry: diagnostic, vaccine, and therapeutic approaches.

Development of molecular biology and understanding structures and functions of various biological molecules and entities allowed to construct various sophisticated tools for different biotechnological, medical, and veterinary applications. One of them is the phage display technology, based on the possibility to create specific bacteriophages bearing fusion genes, which code for fusion proteins consisting of a phage coat protein and a peptide of any amino acid sequence. Such proteins retain their biological functions as structural elements of phage virions while exposing foreign peptide sequences on their surfaces. Genetic manipulations allow to construct phage display libraries composed of billions of variants of exposed peptides; such libraries can be used to select peptides of desired features. Although the phage display technology has been widely used in biotechnology and medicine, its applications in veterinary and especially in poultry science were significantly less frequent. Nevertheless, many interesting discoveries have been reported also in the latter field, providing evidence for a possibility of effective applications of phage display-related methods in developing novel diagnostic tools, new vaccines, and innovative potential therapies dedicated to poultry. Especially, infectious diseases caused by avian viruses, bacteria, and unicellular eukaryotic parasites were investigated in this field. These studies are summarized and discussed in this review, with presentation of various possibilities provided by different phage display systems in development of useful and effective products facilitating management of the problem of infectious diseases of poultry.

Self-assembling protein nanoparticles and virus like particles correctly display β-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal β-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the β-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Development of a synthetic nanoparticle vaccine presenting the HIV-1 envelope glycoprotein

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in Escherichia coli and purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140.

Magnetically Aligned Lipid Bilayers with High Cholesterol for Solid-State NMR of Membrane Proteins.

Phospholipid bicelles are valuable membrane model systems to study membrane proteins by NMR and other physicochemical techniques. The range of bicelle compositions that are compatible with uniaxial alignment of the lipid bilayers in a magnetic field is still limited with regard to the addition of large amounts (>20%) of cholesterol and/or sphingolipids. Here, we demonstrate that n-dodecyl-β-D-melibioside (DDMB), which was recently introduced as a detergent to produce sphingolipid-cholesterol-rich isotropic bicelles for solution NMR studies, can also be used to produce magnetically alignable lipid bilayers with high cholesterol content that are well suited for solid-state NMR of membrane proteins. Remarkably, DDMB enables the preparation of high q bicelles that contain 50% mol cholesterol while retaining their ability to form a stable, well-aligned liquid crystalline bilayer phase in a magnetic field. We show that the intact 46-residue membrane-bound form of Pf1 bacteriophage coat protein and a truncated construct of the membrane protein Vpu from HIV-1 (residues 2-30) in DDMB bicelles are well aligned and undergo fast and uniaxial rotational diffusion about the bilayer normal, similarly to what is observed in other bicelle and macrodisc systems. We also demonstrate a spectroscopic method that measures the increase in the thickness of DMPC bilayers that results from the addition of cholesterol, using the PISA-wheel spectral patterns of trans-membrane helices as a molecular goniometer. For example, we find that the hydrophobic thickness of DMPC bilayers is increased by approximately 2.5 Å in the presence of 35% mol cholesterol.

Viral Ejection Proteins: Mosaically Conserved, Conformational Gymnasts.

Bacterial viruses (or bacteriophages) have developed formidable ways to deliver their genetic information inside bacteria, overcoming the complexity of the bacterial-cell envelope. In short-tailed phages of the Podoviridae superfamily, genome ejection is mediated by a set of mysterious internal virion proteins, also called ejection or pilot proteins, which are required for infectivity. The ejection proteins are challenging to study due to their plastic structures and transient assembly and have remained less characterized than classical components such as the phage coat protein or terminase subunit. However, a spate of recent cryo-EM structures has elucidated key features underscoring these proteins’ assembly and conformational gymnastics that accompany their expulsion from the virion head through the portal protein channel into the host. In this review, we will use a phage-T7-centric approach to critically review the status of the literature on ejection proteins, decipher the conformational changes of T7 ejection proteins in the pre- and post-ejection conformation, and predict the conservation of these proteins in other Podoviridae. The challenge is to relate the structure of the ejection proteins to the mechanisms of genome ejection, which are exceedingly complex and use the host’s machinery.