Mapping the conformational energy landscape of Abl kinase using ClyA nanopore tweezers.

Protein kinases play central roles in cellular regulation by catalyzing the phosphorylation of target proteins. Kinases have inherent structural flexibility allowing them to switch between active and inactive states. Quantitative characterization of kinase conformational dynamics is challenging.

Fast slow folding of an outer membrane porin.

In comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins are surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster resonance energy transfer to report on the folding of fluorescently labeled outer membrane protein G we measured the real-time insertion of a β-barrel membrane protein from an unfolded state. Folding events were rare and fast (<20 ms), occurring immediately upon arrival at the membrane.

Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH.

The outer membrane protein G (OmpG) nanopore is a monomeric β-barrel channel consisting of seven flexible extracellular loops. Its most flexible loop, loop 6, can be used to host high-affinity binding ligands for the capture of protein analytes, which induces characteristic current patterns for protein identification. At acidic pH, the ability of OmpG to detect protein analytes is hampered by its tendency toward the closed state, which renders the nanopore unable to reveal current signal changes induced by bound analytes.

Tuning Protein Discrimination Through Altering the Sampling Interface Formed between the Analyte and the OmpG Nanopore.

Nanopore sensors capable of distinguishing homologous protein analytes are highly desirable tools for proteomics research and disease diagnostics. Recently, an engineered outer membrane protein G (OmpG) nanopore with a high-affinity ligand attached to a gating loop 6 showed specificity for distinguishing homologous proteins in complex mixtures. Here, we report the development of OmpG nanopores with the other six loops used as the anchoring point to host an affinity ligand for protein sensing.

A pH-independent quiet OmpG pore with enhanced electrostatic repulsion among the extracellular loops.

Membrane protein pores have emerged as powerful nanopore sensors for single-molecule detection. OmpG, a monomeric nanopore, is comprised of fourteen β-strands connected by seven flexible extracellular loops. The OmpG nanopore exhibits pH-dependent gating as revealed by planar lipid bilayer studies.

Simulation of pH-Dependent, Loop-Based Membrane Protein Gating Using Pretzel.

Bacterial porins often exhibit ion conductance and gating behavior which can be modulated by pH. However, the underlying control mechanism of gating is often complex, and direct inspection of the protein structure is generally insufficient for full mechanistic understanding. Here we describe Pretzel, a computational framework that can effectively model loop-based gating events in membrane proteins.

A Selective Activity-Based Approach for Analysis of Enzymes with an OmpG Nanopore.

Many enzymatic activity assays are based on either (1) identifying and quantifying the enzyme with methods such as western blot or enzyme-linked substrate assay (ELISA) or (2) quantifying the enzymatic reaction by monitoring the changing levels of either product or substrate. We have generated an outer membrane protein G (OmpG)-based nanopore approach to distinguish enzyme identity as well as analyze the enzyme's catalytic activity. Here, we engineered an OmpG nanopore with a peptide cut site inserted into one of its loops to detect proteolytic behavior.

Protein Analyte Sensing with an Outer Membrane Protein G (OmpG) Nanopore.

Nanopore sensing is a powerful lab-on-a-chip technique that allows for the analysis of biomarkers present in small sample sizes. In general, nanopore clogging and low detection accuracy arise when the sample becomes more and more complex such as in blood or lysate. To address this, we developed an OmpG nanopore that distinguishes among not only different proteins in a mixture but also protein homologs.

A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates.

Caspases are an important protease family that coordinate inflammation and programmed cell death. Two closely related caspases, caspase-3 and caspase-7, exhibit largely overlapping substrate specificities. Assessing their proteolytic activities individually has therefore proven extremely challenging.

Protein Motion and Configurations in a Form-Fitting Nanopore: Avidin in ClyA.

We probe the molecular dynamics and states of an avidin protein as it is captured and trapped in a voltage-biased cytolysin A nanopore using time-resolved single-molecule electrical conductance signals. The data for very large numbers of single-molecule events are analyzed and presented by a new method that provides clear visual insight into the molecular scale processes. Avidin in cytolysin A has surprisingly rich conductance spectra that reveal transient and more permanently trapped protein configurations in the pore and how they evolve into one another.

Disruption of the open conductance in the β-tongue mutants of Cytolysin A.

Cytolysin A (ClyA) is a water-soluble alpha pore-forming toxin that assembles to form an oligomeric pore on host cell membranes. The ClyA monomer possesses an α-helical bundle with a β-sheet subdomain (the β-tongue) previously believed to be critical for pore assembly and/or insertion. Oligomerization of ClyA pores transforms the β-tongue into a helix-turn-helix that embeds into the lipid bilayer.

Mechanism of OmpG pH-Dependent Gating from Loop Ensemble and Single Channel Studies.

Outer membrane protein G (OmpG) from Escherichia coli has exhibited pH-dependent gating that can be employed by bacteria to alter the permeability of their outer membranes in response to environmental changes. We developed a computational model, Protein Topology of Zoetic Loops (Pretzel), to investigate the roles of OmpG extracellular loops implicated in gating. The key interactions predicted by our model were verified by single-channel recording data.

Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif.

β-Barrelmembrane proteins (βMPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, βMPs have great potential as nanosensors for single-molecule detection. However, natural βMPs currently employed have inflexible biophysical properties and are limited in their pore geometry, hindering their applications in sensing molecules of different sizes and properties.

Tuning the selectivity and sensitivity of an OmpG nanopore sensor by adjusting ligand tether length.

We have previously shown that a biotin ligand tethered to the rim of an OmpG nanopore can be used to detect biotin-binding proteins. Here, we investigate the effect of the length of the polyethylene glycol tether on the nanopore's sensitivity and selectivity. When the tether length was increased from 2 to 45 ethylene repeats, sensitivity decreased substantially for a neutral protein streptavidin and slightly for a positively charged protein (avidin).

Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore.

Outer membrane protein G is a monomeric β-barrel porin that has seven flexible loops on its extracellular side. Conformational changes of these labile loops induce gating spikes in current recordings that we exploited as the prime sensing element for protein detection. The gating characteristics, open probability, frequency, and current decrease, provide rich information for analyte identification.

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms.

The flexible loops decorating the entrance of OmpG nanopore move dynamically during ionic current recording. The gating caused by these flexible loops changes when a target protein is bound. The gating is characterized by parameters including frequency, duration, and open-pore current, and these features combine to reveal the identity of a specific analyte protein.

Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.

Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore.

A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.

Cytolysin A (ClyA) is an α-pore forming toxin from pathogenic Escherichia coli (E. coli) and Salmonella enterica. Here, we report that E.