Oxytocin, a neurohormone peptide, is implicated in various physiological processes, such as functions related to social behavior, reproduction, and emotional regulation. Recently, this peptide has been identified as a potential biomarker of human psychiatric disorders. Until now, no analytical tool has been able to routinely detect oxytocin and its variants, which can have different effects on their receptor binding and activity. This work uses an aerolysin nanopore and two mutants to detect oxytocin (OXT) and oxytocin-free acid (OXT-FA) differing by a single amine group. Several oxytocin variants were used to understand the effect of structure and conformational changes of the peptides on the electric signal. We identify two conformations in native conditions, allowing their discrimination. Nanopore mutants showed improved discrimination of these peptides. Molecular dynamics simulation allowed us to rationalize the competition between electroosmosis and electrophoresis on OXT and OXT-FA transport through aerolysin. This work demonstrates the potential for analyzing conformational biomarkers for diagnostic purposes.

There is a current need to develop methods for sensitively detecting peptide biomarkers in complex biofluids to enable early disease detection. Moreover, to our knowledge, no detection method is currently capable of identifying the different conformations of peptide biomarkers differing by a single amino acid, or L- and D-peptide enantiomers. Single-molecule nanopore sensing promises to provide this level of resolution. To identify these differences in a complex biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed.

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Thermophages represent a subset of bacteriophages, viruses that target bacteria, thriving in extreme temperature environments worldwide. Tailed bacteriophages contain a portal protein, serving as a natural DNA translocating channel, located at a single vertex of their viral capsid. These portals play a crucial role in facilitating the pumping of DNA into capsids during virus assembly. Thermophages are valuable reservoirs of thermally stable proteins for use in biotechnology. Notably, successful efforts have been made to harness portal proteins from thermophages, such as portal from bacteriophage G20c, for applications as both biological and hybrid nanopores.

DNA recognition is critical for assembly of double-stranded DNA viruses, in particular for the initiation of packaging the viral genome into the capsid. DNA packaging has been extensively studied for three archetypal bacteriophage systems: cos, pac and phi29. We identified the minimal site within the cos region of bacteriophage HK97 specifically recognised by the small terminase and determined a cryoEM structure for the small terminase:DNA complex. This nonameric circular protein utilizes a previously unknown mechanism of DNA binding. While DNA threads through the central tunnel, unexpectedly, DNA-recognition is generated at its exit by a substructure formed by the N- and C-terminal segments of two adjacent protomers of the terminase which are unstructured in the absence of DNA. Such interaction ensures continuous engagement of the small terminase with DNA, allowing sliding along DNA while simultaneously checking the DNA sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the cos site.

There is a current need to develop methods for the sensitive detection of peptide biomarkers in complex mixtures of molecules, such as biofluids, to enable early disease detection. Moreover, to our knowledge, there is currently no detection method capable of identifying the different conformations of a peptide biomarker differing by a single amino acid. Single-molecule nanopore sensing promises to provide this level of resolution. In order to be able to identify these differences in a biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed. We are interested here in a family of peptide biomarkers, kinins such as bradykinin and des-Arg9 bradykinin, that are involved in many disabling pathologies (allergy, asthma, angioedema, sepsis, or cancer). We show the proof of concept for direct identification of these biomarkers in serum at the single-molecule level using a protein nanopore. Each peptide exhibits two unique electrical signatures attributed to specific conformations in bulk. The same signatures are found in serum, allowing their discrimination and identification in a complex mixture such as biofluid. To extend the utility of our experimental results, we developed a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used semisupervised classification to assign each event type to a specific biomarker at physiological serum concentration. In the future, single-molecule scale analysis of peptide biomarkers using a powerful nanopore coupled with machine learning will facilitate the identification and quantification of other clinically relevant biomarkers from biofluids.

One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

With an increasing global population that is rapidly ageing, our society faces challenges that impact health, environment, and energy demand. With this ageing comes an accumulation of cellular changes that lead to the development of diseases and susceptibility to infections. This impacts not only the health system, but also the global economy. As the population increases, so does the demand for energy and the emission of pollutants, leading to a progressive degradation of our environment. This in turn impacts health through reduced access to arable land, clean water, and breathable air. New monitoring approaches to assist in environmental control and minimize the impact on health are urgently needed, leading to the development of new sensor technologies that are highly sensitive, rapid, and low-cost. Nanopore sensing is a new technology that helps to meet this purpose, with the potential to provide rapid point-of-care medical diagnosis, real-time on-site pollutant monitoring systems to manage environmental health, as well as integrated sensors to increase the efficiency and storage capacity of renewable energy sources. In this review we discuss how the powerful approach of nanopore based single-molecule, or particle, electrical promises to overcome existing and emerging societal challenges, providing new opportunities and tools for personalized medicine, localized environmental monitoring, and improved energy production and storage systems.

The application of nanopores as label-free, single-molecule biosensors for electrical or optical probing of structural features in biomolecules has been widely explored. While biological nanopores (membrane proteins and bacteriophage portal proteins) and solid-state nanopores (thin films and two-dimensional materials) have been extensively employed, the third class of nanopores known as hybrid nanopores, where an artificial membrane substitutes the organic support membrane of proteins, has been only sparsely studied due to challenges in implementation. G20c portal protein contains a natural DNA pore that is used by viruses for filling their capsid with viral genomic DNA. We have previously developed a lipid-free hybrid nanopore by "corking" the G20c portal protein into a SiNx nanopore. Herein, we demonstrate that through chemical functionalization of the synthetic nanopore, covalent linkage between the solid-state pore and the G20c portal protein considerably improves the hybrid pore stability, lifetime, and voltage resilience. Moreover, we demonstrate electric-field-driven and motor protein-mediated transport of DNA molecules through this hybrid nanopore. Our integrated protein/solid-state device can serve as a robust and durable framework for sensing and sequencing at high voltages, potentially providing higher resolution, higher signal-to-noise ratio, and higher throughput compared to the more conventional membrane-embedded protein platforms.

Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the "3-bases-in, 3-bases-out" conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123-139, which may serve as a valuable epitope for surveillance and diagnostics.

Double stranded DNA bacteriophages selectively package the new copies of their genomic DNA into preassembled protein capsid shells. The newly synthesized viral DNA is recognized by a specialized protein complex made up of multiple copies of the DNA recognition protein (small terminase) along with one or more subunits of the motor protein (large terminase). These proteins act together to place one end of the genomic DNA inside the DNA translocation motor assembled onto a unique pore in the capsid shell ready for packaging.

Site-specific, reproducible, and uniform-orientation protein immobilization on an inorganic surface while preserving protein conformation and activity is of wide interest for applications in biosensing, protein microarrays, and enzymology, among others. Successful immobilization requires understanding of material's surface chemistry, protein properties, and nature of their interaction to eliminate non-specific binding. Portal protein is a naturally occurring pore (biological nanopore) and part of the bacteriophage packaging machine that pumps the viral genome inside its capsid. In this work, we have explored different approaches to immobilize G20c portal protein from double-stranded bacteriophage G20c into a thin (∼30 nm) silicon nitride (SiNx) membrane embedded in a silicon chip. Desired orientation of the immobilized protein is achieved by maintaining a voltage bias across the membrane to electrokinetically drive a single protein into the synthetic nanopore. The nanopore geometry dictates a predominant favorable protein orientation while the portal preserves its conformation, as indicated by ion current measurements through the “hybrid nanopore”. Application of nanopores (both synthetic and biological) as label-free, single-molecule biosensors for electrical and/or optical probing of structural features in biomolecules have been widely explored. Confirmed by our single-molecule electrical sensing results, our hybrid nanopore system provides mechanically robust and chemically compatible synthetic protein framework, superior to its natural counterparts such as organic membranes (lipid bilayer, for example), and exploits tunable and engineerable characteristics of thermostable G20c portal protein rendering an active, high-resolution biomolecule sensing platform. We demonstrate here through chemical functionalization of synthetic nanopores and/or engineering G20c portal protein assemblies, a protein nanopore chemically linked to a synthetic nanopore, rendering considerably improved protein stability, sensing lifetime, and signal-to-noise ratio compared to our previous hybrid system. This development will be widely applicable to coupled nanopore sensor arrangements such as electro-optical and electro-pressure systems.

Nipah virus is a highly pathogenic zoonotic RNA virus, causing fatal encephalitis in humans. Like other negative-strand RNA viruses including Ebola and measles, its genome is wrapped by the nucleocapsid (N) protein forming a helical assembly. Here we report the CryoEM structure of the Nipah nucleocapsid protein-RNA assembly, at near atomic resolution. The N protein wraps the RNA genome with a periodicity of six nucleotides per protomer, around the outer edge of the helical assembly, in common with other paramyxoviruses. This structure uncovers details of the nucleocapsid assembly, demonstrating the role of the N-terminal arm of the N protein in the formation of the helical assembly and revealing details of the sequence-independent coordination of RNA binding in the “3-bases-in, 3-bases-out” conformation. CryoEM analysis also reveals formation of clam-shaped assemblies of the N-protein, mediated by intersubunit interactions involving several N protein loop regions.

Flaviviruses are single-stranded positive sense RNA viruses.Several members of this family, such as Dengue, Yellow Fever, and Zika viruses, are associated with important human diseases.Flaviviral non-structural protein 3 (NS3) helicase participates in RNA unwinding and capping, and is crucial for viral replication [1,2].X-ray structures for protein complexes with a polynucleotide and/or NTP analogs have been reported for several Flaviviral NS3 helicases.However, the mechanism of coupling NTP hydrolysis with RNA translocation remains unclear.In this study, we determined the first crystal structure for the Zika virus NS3 helicase complex with an ssRNA segment containing 5'-phosphate.Notably, the presence of the 5'-phosphate induces significant remodeling of the interactions with the 5'-nucleotide of RNA, unveiling the breadth of structural changes that can occur during RNA unwinding.Additionally, we determined the X-ray structures of the NS3 helicase in several different functional states of NTP hydrolysis, including the pre-hydrolysis, transition state and post-hydrolysis.Structural observations indicate how a catalytically important loop, that is involved in ATP hydrolysis, can mediate coupling with RNA translocation.Structural data are substantiated by NMR analysis of the NS3 helicase transition state complex, which demonstrate that the presence of RNA enhances the transition state formation.Taken together, our data clarify the mechanism for RNA unwinding that is applicable to all Flaviviridae family NS3 helicases.

Nanometer-sized pores (nanopores) have emerged as a novel single-molecule detection technique to probe biomolecules (protein, DNA, and RNA) electrically and/or optically whilst being threaded into the pore. Since then, naturally-occurring pores known as biological nanopores (membrane proteins and bacteriophage portal proteins) along with synthetically-assembled nanopores known as solid-state nanopores have been employed to study various biomolecular characteristics. A third class of nanopores, known as hybrid nanopores, exploits the robust framework and mechanical stability of SS nanopores with atomically reproducible and tunable characteristics of biological nanopores. Biological nanopores supported by robust synthetic membrane eliminates fragility of lipid bilayers and arbitrary geometry of SS nanopores. Our group recently demonstrated the lipid insertion and stable formation of hydrophilic G20C portal protein transmembrane channels, derived from the G20C thermostable virus. Herein, we demonstrate the improved performance of our system by substituting the lipid bilayer support of the protein by thin SiNx free-standing membrane and making further modifications to chemically fix the protein on the membrane support. Finally, we report the application of our hybrid system in nanopore-based sensing of biomolecules.

Nanopore-based sensors are advancing the sensitivity and selectivity of single-molecule detection in molecular medicine and biotechnology. Current electrical sensing devices are based on either membrane protein pores supported in planar lipid bilayers or solid-state (SS) pores fabricated in thin metallic membranes. While both types of nanosensors have been used in a variety of applications, each has inherent disadvantages that limit its use. Hybrid nanopores, consisting of a protein pore supported within a SS membrane, combine the robust nature of SS membranes with the precise and simple engineering of protein nanopores. We demonstrate here a novel lipid-free hybrid nanopore comprising a natural DNA pore from a thermostable virus, electrokinetically inserted into a larger nanopore supported in a silicon nitride membrane. The hybrid pore is stable and easy to fabricate, and, most importantly, exhibits low peripheral leakage allowing sensing and discrimination among different types of biomolecules.

In eukaryotes, several "hub" proteins integrate signals from different interacting partners that bind through intrinsically disordered regions. The 14-3-3 protein hub, which plays wide-ranging roles in cellular processes, has been linked to numerous human disorders and is a promising target for therapeutic intervention. Partner proteins usually bind via insertion of a phosphopeptide into an amphipathic groove of 14-3-3. Structural plasticity in the groove generates promiscuity allowing accommodation of hundreds of different partners. So far, accurate structural information has been derived for only a few 14-3-3 complexes with phosphopeptide-containing proteins and a variety of complexes with short synthetic peptides. To further advance structural studies, here we propose a novel approach based on fusing 14-3-3 proteins with the target partner peptide sequences. Such chimeric proteins are easy to design, express, purify and crystallize. Peptide attachment to the C terminus of 14-3-3 via an optimal linker allows its phosphorylation by protein kinase A during bacterial co-expression and subsequent binding at the amphipathic groove. Crystal structures of 14-3-3 chimeras with three different peptides provide detailed structural information on peptide-14-3-3 interactions. This simple but powerful approach, employing chimeric proteins, can reinvigorate studies of 14-3-3/phosphoprotein assemblies, including those with challenging low-affinity partners, and may facilitate the design of novel biosensors.

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism.

Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2β/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts.