Biology subjects

Parent, K. N.

Publications and source records attributed to Parent, K. N..

10 recordsLinked to original sources

bioRxiv2026

Screening metatranscriptomes for ultrastable RNA secondary structures reveals hidden bacteriophages and novel capsid nanomaterials

Metatranscriptomics has transformed our view of RNA bacteriophage diversity, revealing vast numbers of single-stranded RNA (ssRNA) phages whose protein capsids can be engineered for biotechnology applications. However, many ssRNA phages remain hidden from current detection methods, which require protein-level similarity to known phages. Here we show that RNA structure provides an additional signal for the detection of ssRNA phages in metatranscriptomes, including hidden phages missed by prior protein-based methods. By computationally folding each contig and screening for exceptionally stable RNA secondary structures, we find evidence of thousands of previously unrecognized phages encoding novel coat proteins. We express a library of 12,000 such coat proteins in E. coli and find that most assemble into nuclease-resistant capsids. We determine the 3D structure of one such capsid by cryo-electron microscopy and demonstrate that it can be disassembled and reassembled in vitro to package heterologous RNA--a key step toward repurposing these particles as RNA delivery vehicles. We compile the newly discovered ssRNA phages with previously known ones into a database that contains sequence and structural information for over 460,000 unique RNA molecules and over 100,000 distinct coat proteins, providing a comprehensive resource for microbiology and nanomaterials research.

biochemistry↗

bioRxiv2026

Encapsulation in a bacterial microcompartment shell improves thermal stability of a glycolytic enzyme

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMCs) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62{degrees}C.

synthetic biology↗

bioRxiv2025

Structures of two LarA-like nickel-pincer nucleotide cofactor-utilizing enzymes with a single catalytic histidine residue

The nickel pincer nucleotide (NPN) cofactor catalyzes the racemization/epimerization of -hydroxy acids in enzymes of the LarA family. The established proton-coupled hydride transfer mechanism requires two catalytic histidine residues that alternately act as general acids and general bases. Notably, however, a fraction of LarA homologs (LarAHs) lack one of the active site histidine residues, replacing it with an asparaginyl side chain that cannot participate in acid/base catalysis. Here, we investigated two such LarAHs and solved their cryo-electron microscopic structures with and without loaded NPN cofactor, respectively. The structures revealed a consistent octameric assembly that is unprecedented in the LarA family and unveiled a new set of active site residues that likely recognize and process substrates differently from those of the well-studied LarAHs. Genomic context analysis suggested their potential involvement in carbohydrate metabolism. Together, these findings lay the groundwork for expanding the breadth of reactions and the range of mechanisms of LarA enzymes.

biochemistry↗

bioRxiv2025

Measuring the selective packaging of RNA molecules by viral coat proteins in cells

Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules in E. coli. In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known TR stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes. Significance StatementBacteriophage MS2 packages its RNA genome into protective protein shells called capsids while excluding nearly all host-cell RNA. Engineering synthetic capsids with similar selectivity could enable a broad range of RNA-based technologies, including CRISPR gene editing systems, mRNA vaccines, and other emerging RNA-based therapeutics. Our study shows that selective packaging in MS2 is not dictated by a single, high-affinity RNA-protein interaction but instead emerges from the collective interactions of multiple coat proteins and an ensemble of stem-loops distributed across the RNA molecule. By establishing these collective interactions as the basis of selectivity, our findings provide a foundation for engineering synthetic capsids capable of selectively packaging target RNAs for next-generation RNA-based technologies.

biophysics↗

bioRxiv2023

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, PLE, remodels the capsid it has been predicted to steal from the phage ICP1 (1). 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 (2). 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.

microbiology↗

bioRxiv2022

Host range expansion of Shigella phage Sf6 evolves through the dual roles of its tailspike

The first critical step in a viruss infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable co-evolution and host range switching or expansion. Like many bacterial viruses, bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host, Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) structure of S. flexneri, then binds to either outer membrane protein (Omp) A or OmpC. This phage typically infects serotype Y strains but can also form small, turbid plaques on serotype 2a2 with greatly reduced plating efficiency, suggesting inefficient infection. To examine the interactions between Sf6 and this sub-optimal host, phage were experimentally evolved using mixed populations of S. flexneri serotypes Y and 2a2. The recovered mutants could infect serotype 2a2 with greater efficiency than the ancestral Sf6, forming clear plaques on both serotypes. All mutations mapped to two distinct regions of the tailspike protein: 1) adjacent to, but not part of, the LPS binding site near the N-terminus; and 2) at the distal, C-terminal tip of the protein. Rather than weak interactions between the Sf6 tailspike and 2a2 O-antigen, LPS of this serotype appears to inhibit infection by binding the wild-type particles more strongly, effectively removing them from the environment. These mutations reduce the inhibitory effect by either reducing electrostatic interactions with the O-antigen or increasing reliance on the Omp secondary receptors.

microbiology↗

bioRxiv2021

A Broadly Conserved Deoxycytidine Deaminase Protects Bacteria from Phage Infection

The El Tor biotype of Vibrio cholerae is responsible for perpetuating the longest cholera pandemic in recorded history (1961-current). The genomic islands VSP-1 and -2 are two understudied genetic features that distinguish El Tor from previous pandemics. To understand their utility, we calculated the co-occurrence of VSP genes across bacterial genomes. This analysis predicted the previously uncharacterized vc0175, herein renamed deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, a cyclic GMP-AMP synthase involved in phage defense. DcdV consists of two domains, a P-loop kinase and a deoxycytidylate deaminase, that are required for the deamination of dCTP and dCMP, inhibiting phage predation by corrupting cellular nucleotide concentrations. Additionally, DcdV is post-translationally inhibited by a unique noncoding RNA encoded 5 of the dcdV locus. DcdV homologs are conserved in bacteria and eukaryotes and our results identify V. cholerae DcdV as the founding member of a previously undescribed bacterial phage defense system.

microbiology↗

bioRxiv2020

The Mitochondrial Permeability Transition Phenomenon Elucidated by Cryo-EM Reveals the Genuine Impact of Calcium Overload on Mitochondrial Structure and Function

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves inner membrane architecture despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

cell biology↗

bioRxiv2019

Boiling Acid Mimics Intracellular Giant Virus Genome Release

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four metastable infection intermediates in Samba virus (lineage A Mimiviridae) as visualized by cryo-EM, cryo-ET, and SEM. Each of these four intermediates reflects a stage that occurs in vivo. We show that these genome release stages are conserved in other, diverse giant viruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved amongst disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

biophysics↗

bioRxiv2018

How phage L capsid decoration protein distinguishes between nearly identical binding sites on an icosahedral virion

The major coat proteins of dsDNA tailed phages and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary "decoration" (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. Lastly, the NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

biophysics↗