Gating of ß-Barrel Protein Pores, Porins, and Channels: An Old Problem with New Facets.

ß barrels are ubiquitous proteins in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. These transmembrane proteins (TMPs) execute a wide variety of tasks. For example, they can serve as transporters, receptors, membrane-bound enzymes, as well as adhesion, structural, and signaling elements. In addition, multimeric ß barrels are common structural scaffolds among many pore-forming toxins. Significant progress has been made in understanding the functional, structural, biochemical, and biophysical features of these robust and versatile proteins. One frequently encountered fundamental trait of all ß barrels is their voltage-dependent gating. This process consists of reversible or permanent conformational transitions between a large-conductance, highly permeable open state and a low-conductance, solute-restrictive closed state. Several intrinsic molecular mechanisms and environmental factors modulate this universal property of ß barrels. This review article outlines the typical signatures of voltage-dependent gating. Moreover, we discuss recent developments leading to a better qualitative understanding of the closure dynamics of these TMPs.

Current noise of a protein-selective biological nanopore.

1/f current noise is ubiquitous in protein pores, porins, and channels. We have previously shown that a protein-selective biological nanopore with an external protein receptor can function as a 1/f noise generator when a high-affinity protein ligand is reversibly captured by the receptor. Here, we demonstrate that the binding affinity and concentration of the ligand are key determinants for the nature of current noise. For example, 1/f was absent when a protein ligand was reversibly captured at a much lower concentration than its equilibrium dissociation constant against the receptor. Furthermore, we also analyzed the composite current noise that resulted from mixtures of low-affinity and high-affinity ligands against the same receptor. This study highlights the significance of protein recognition events in the current noise fluctuations across biological membranes.

Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions.

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

Protein Ligand-Induced Amplification in the 1/f Noise of a Protein-Selective Nanopore.

Previous studies of transmembrane protein channels have employed noise analysis to examine their statistical current fluctuations. In general, these explorations determined a substrate-induced amplification in the Gaussian white noise of these systems at a low-frequency regime. This outcome implies a lack of slowly appearing fluctuations in the number and local mobility of diffusing charges in the presence of channel substrates. Such parameters are among the key factors in generating a low-frequency 1/f noise. Here, we show that a protein-selective biological nanopore exhibits a substrate-induced amplification in the 1/f noise. The modular composition of this biological nanopore includes a hydrophilic transmembrane protein pore fused to a water-soluble binding protein on its extramembranous side. In addition, this protein nanopore shows an open substate populated by a high-frequency current noise because of the flickering of an engineered polypeptide adaptor at the tip of the pore. However, the physical association of the protein ligand with the binding domain reversibly switches the protein nanopore from a high-frequency noise substate into a quiet substate. In the absence of the protein ligand, our nanopore shows a low-frequency white noise. Remarkably, in the presence of the protein ligand, an amplified low-frequency 1/f noise was detected in a ligand concentration-dependent fashion. This finding suggests slowly occurring equilibrium fluctuations in the density and local mobility of charge carriers under these conditions. Furthermore, we report that the excess in 1/f noise is generated by reversible switches between the noisy ligand-released substate and the quiet ligand-captured substate. Finally, quantitative aspects of the low-frequency 1/f noise are in accord with theoretical predictions of the current noise analysis of protein channel-ligand interactions.

Cyclic Activity of an Osmotically Stressed Liposome in a Finite Hypotonic Environment.

A lipid vesicle, or simply called a liposome, represents a synthetic compartment for the examination of transmembrane transport and signaling phenomena. Yet, a liposome is always subjected to size and shape fluctuations due to local and global imbalance of internal and external osmotic pressures. Here, we show that an osmotically stressed liposome placed within a hypotonic spherical bath undergoes cyclic dynamics described by a periodic sequence of swelling and relaxation phases. These two phases are interfaced by the appearance of a transient transmembrane pore through which chemical delivery occurs. An analytical model was formulated for the recurrent differential equations that convey the time-dependent swelling phase of a pulsatory liposome during individual cycles. We demonstrate that the time-dependent swelling phases of the last several cycles of a pulsatory liposome are strongly dependent on the size of the external bath. Furthermore, decreasing the size of the hypotonic medium reduces the number of cycles of a pulsatory liposome. Comparisons and contrasts of an infinite hypotonic bath with finite external baths of varying radii are discussed.

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores.

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ∼8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric ß-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

Global redesign of a native ß-barrel scaffold.

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded ß-barrel protein into a large-conductance protein pore. Here, we show that this redesigned ß-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric ß-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.

p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.

Substrate specificity within a family of outer membrane carboxylate channels.

Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

Toward understanding the outer membrane uptake of small molecules by Pseudomonas aeruginosa.

Because small molecules enter Gram-negative bacteria via outer membrane (OM) channels, understanding OM transport is essential for the rational design of improved and new antibiotics. In the human pathogen Pseudomonas aeruginosa, most small molecules are taken up by outer membrane carboxylate channel (Occ) proteins, which can be divided into two distinct subfamilies, OccD and OccK. Here we characterize substrate transport mediated by Occ proteins belonging to both subfamilies. Based on the determination of the OccK2-glucuronate co-crystal structure, we identify the channel residues that are essential for substrate transport. We further show that the pore regions of the channels are rigid in the OccK subfamily and highly dynamic in the OccD subfamily. We also demonstrate that the substrate carboxylate group interacts with central residues of the basic ladder, a row of arginine and lysine residues that leads to and away from the binding site at the channel constriction. Moreover, the importance of the basic ladder residues corresponds to their degree of conservation. Finally, we apply the generated insights by converting the archetype of the entire family, OccD1, from a basic amino acid-specific channel into a channel with a preference for negatively charged amino acids.

Does the lipid environment impact the open-state conductance of an engineered ß-barrel protein nanopore?

Using rational membrane protein design, we were recently able to obtain a ß-barrel protein nanopore that was robust under an unusually broad range of experimental circumstances. This protein nanopore was based upon the native scaffold of the bacterial ferric hydroxamate uptake component A (FhuA) of Escherichia coli. In this work, we expanded the examinations of the open-state current of this engineered protein nanopore, also called FhuA ΔC/Δ4L, employing an array of lipid bilayer systems that contained charged and uncharged as well as conical and cylindrical lipids. Remarkably, systematical single-channel analysis of FhuA ΔC/Δ4L indicated that most of its biophysical features, such as the unitary conductance and the stability of the open-state current, were not altered under the conditions tested in this work. However, electrical recordings at high transmembrane potentials revealed that the presence of conical phospholipids within the bilayer catalyzes the first, stepwise current transition of the FhuA ΔC/Δ4L protein nanopore to a lower-conductance open state. This study reinforces the stability of the open-state current of the engineered FhuA ΔC/Δ4L protein nanopore under various experimental conditions, paving the way for further critical developments in biosensing and molecular biomedical diagnosis.

Overlapping characteristics of weak interactions of two transcriptional regulators with WDR5.

The WD40 repeat protein 5 (WDR5) is a nuclear hub that critically influences gene expression by interacting with transcriptional regulators. Utilizing the WDR5 binding motif (WBM) site, WDR5 interacts with the myelocytomatosis (MYC), an oncoprotein transcription factor, and the retinoblastoma-binding protein 5 (RbBP5), a scaffolding element of an epigenetic complex. Given the clinical significance of these protein-protein interactions (PPIs), there is a pressing necessity for a quantitative assessment of these processes. Here, we use biolayer interferometry (BLI) to examine interactions of WDR5 with consensus peptide ligands of MYC and RbBP5. We found that both interactions exhibit relatively weak affinities arising from a fast dissociation process. Remarkably, live-cell imaging identified distinctive WDR5 localizations in the absence and presence of full-length binding partners. Although WDR5 tends to accumulate within nucleoli, WBM-mediated interactions with MYC and RbBP5 require their localization outside nucleoli. We utilize fluorescence resonance energy transfer (FRET) microscopy to confirm these weak interactions through a low FRET efficiency of the MYC-WDR5 and RbBP5-WDR5 complexes in living cells. In addition, we evaluate the impact of peptide and small-molecule inhibitors on these interactions. These outcomes form a fundamental basis for further developments to clarify the multitasking role of the WBM binding site of WDR5.

Cation selectivity is a conserved feature in the OccD subfamily of Pseudomonas aeruginosa.

To achieve the uptake of small, water-soluble nutrients, Pseudomonas aeruginosa, a pathogenic Gram-negative bacterium, employs substrate-specific channels located within its outer membrane. In this paper, we present a detailed description of the single-channel characteristics of six members of the outer membrane carboxylate channel D (OccD) subfamily. Recent structural studies showed that the OccD proteins share common features, such as a closely related, monomeric, 18-stranded ß-barrel conformation and large extracellular loops, which are folded back into the channel lumen. Here, we report that the OccD proteins displayed single-channel activity with a unitary conductance covering an unusually broad range, between 20 and 670pS, as well as a diverse gating dynamics. Interestingly, we found that cation selectivity is a conserved trait among all members of the OccD subfamily, bringing a new distinction between the members of the OccD subfamily and the anion-selective OccK channels. Conserved cation selectivity of the OccD channels is in accord with an increased specificity and selectivity of these proteins for positively charged, carboxylate-containing substrates.

Multiplexed imaging for probing RAS-RAF interactions in living cells.

GTP-bound RAS interacts with its protein effectors in response to extracellular stimuli, leading to chemical inputs for downstream pathways. Significant progress has been made in measuring these reversible protein-protein interactions (PPIs) in various cell-free environments. Yet, acquiring high sensitivity in heterogeneous solutions remains challenging. Here, using an intermolecular fluorescence resonance energy transfer (FRET) biosensing approach, we develop a method to visualize and localize HRAS-CRAF interactions in living cells. We demonstrate that the EGFR activation and the HRAS-CRAF complex formation can be concurrently probed in a single cell. This biosensing strategy discriminates EGF-stimulated HRAS-CRAF interactions at the cell and organelle membranes. In addition, we provide quantitative FRET measurements for assessing these transient PPIs in a cell-free environment. Finally, we prove the utility of this approach by showing that an EGFR-binding compound is a potent inhibitor of HRAS-CRAF interactions. The outcomes of this work form a fundamental basis for further explorations of the spatiotemporal dynamics of various signaling networks.

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses.

Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors' optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.

A generalizable nanopore sensor for highly specific protein detection at single-molecule precision.

Protein detection has wide-ranging implications in molecular diagnostics. Substantial progress has been made in protein analytics using nanopores and the resistive-pulse technique. Yet, a long-standing challenge is implementing specific interfaces for detecting proteins without the steric hindrance of the pore interior. Here, we formulate a class of sensing elements made of a programmable antibody-mimetic binder fused to a monomeric protein nanopore. This way, such a modular design significantly expands the utility of nanopore sensors to numerous proteins while preserving their architecture, specificity, and sensitivity. We prove the power of this approach by developing and validating nanopore sensors for protein analytes that drastically vary in size, charge, and structural complexity. These analytes produce unique electrical signatures that depend on their identity and quantity and the binder-analyte assembly at the nanopore tip. The outcomes of this work could impact biomedical diagnostics by providing a fundamental basis for biomarker detection in biofluids.

Inspection of the engineered FhuA ΔC/Δ4L protein nanopore by polymer exclusion.

Extensive engineering of protein nanopores for biotechnological applications using native scaffolds requires further inspection of their internal geometry and size. Recently, we redesigned ferric hydroxamate uptake component A (FhuA), a 22-ß-stranded protein containing an N-terminal 160-residue cork domain (C). The cork domain and four large extracellular loops (4L) were deleted to obtain an unusually stiff engineered FhuA ΔC/Δ4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of FhuA ΔC/Δ4L. When this nanopore was reconstituted into a synthetic planar lipid bilayer, addition of poly(ethylene glycols) produced modifications in the single-channel conductance, allowing for the evaluation of the nanopore diameter. Here, we report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance smaller than the trans entrance, in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Further experiments with impermeable dextran polymers indicated an average internal diameter of ~2.4 nm, a conclusion we arrived at based upon the polymer-induced alteration of the access resistance contribution to the nanopore's total resistance. Molecular insights inferred from this work represent a platform for future protein engineering of FhuA that will be employed for specific tasks in biotechnological applications.

An outer membrane protein undergoes enthalpy- and entropy-driven transitions.

ß-Barrel membrane proteins often fluctuate among various open substates, yet the nature of these transitions is not fully understood. Using temperature-dependent, single-molecule electrophysiology analysis, along with rational protein design, we show that OccK1, a member of the outer membrane carboxylate channel from Pseudomonas aeruginosa, features a discrete gating dynamics comprising both enthalpy-driven and entropy-driven current transitions. OccK1 was chosen for the analysis of these transitions, because it is a monomeric transmembrane ß-barrel of a known high-resolution crystal structure and displays three distinguishable, time-resolvable open substates. Native and loop-deletion OccK1 proteins showed substantial changes in the activation enthalpies and entropies of the channel transitions, but modest alterations in the equilibrium free energies, confirming that the system never departs from equilibrium. Moreover, some current fluctuations of OccK1 indicated a counterintuitive, negative activation enthalpy, which was compensated by a significant decrease in the activation entropy. Temperature scanning of the single-channel properties of OccK1 exhibited a thermally induced switch of the energetically most favorable open substate at the lowest examined temperature of 4 °C. Therefore, such a semiquantitative assessment of the current fluctuation dynamics not only demonstrates the complexity of channel gating but also reveals distinct functional traits of a ß-barrel outer membrane protein under different temperature circumstances.

OccK channels from Pseudomonas aeruginosa exhibit diverse single-channel electrical signatures but conserved anion selectivity.

Pseudomonas aeruginosa is a Gram-negative bacterium that utilizes substrate-specific outer membrane (OM) proteins for the uptake of small, water-soluble nutrients employed in the growth and function of the cell. In this paper, we present for the first time a comprehensive single-channel examination of seven members of the OM carboxylate channel K (OccK) subfamily. Recent biochemical, functional, and structural characterization of the OccK proteins revealed their common features, such as a closely related, monomeric, 18-stranded ß-barrel conformation with a kidney-shaped transmembrane pore and the presence of a basic ladder within the channel lumen. Here, we report that the OccK proteins exhibited fairly distinct unitary conductance values, in a much broader range than previously expected, which includes low (~40-100 pS) and medium (~100-380 pS) conductance. These proteins showed diverse single-channel dynamics of current gating transitions, revealing one-open substate (OccK3), two-open substate (OccK4-OccK6), and three-open substate (OccK1, OccK2, and OccK7) kinetics with functionally distinct conformations. Interestingly, we discovered that anion selectivity is a conserved trait among the members of the OccK subfamily, confirming the presence of a net pool of positively charged residues within their central constriction. Moreover, these results are in accord with an increased specificity and selectivity of these protein channels for negatively charged, carboxylate-containing substrates. Our findings might ignite future functional examinations and full atomistic computational studies for unraveling a mechanistic understanding of the passage of small molecules across the lumen of substrate-specific, ß-barrel OM proteins.