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.

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.

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.

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.

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.

Convergent Alterations of a Protein Hub Produce Divergent Effects within a Binding Site.

Progress in tumor sequencing and cancer databases has created an enormous amount of information that scientists struggle to sift through. While several research groups have created computational methods to analyze these databases, much work still remains in distinguishing key implications of pathogenic mutations. Here, we describe an approach to identify and evaluate somatic cancer mutations of WD40 repeat protein 5 (WDR5), a chromatin-associated protein hub. This multitasking protein maintains the functional integrity of large multi-subunit enzymatic complexes of the six human SET1 methyltransferases. Remarkably, the somatic cancer mutations of WDR5 preferentially distribute within and around an essential cavity, which hosts the WDR5 interaction (Win) binding site. Hence, we assessed the real-time binding kinetics of the interactions of key clustered WDR5 mutants with the Win motif peptide ligands of the SET1 family members (SET1Win). Our measurements highlight that this subset of mutants exhibits divergent perturbations in the kinetics and strength of interactions not only relative to those of the native WDR5 but also among various SET1Win ligands. These outcomes could form a fundamental basis for future drug discovery and other developments in medical biotechnology.

Interplay of Affinity and Surface Tethering in Protein Recognition.

Surface-tethered ligand-receptor complexes are key components in biological signaling and adhesion. They also find increasing utility in single-molecule assays and biotechnological applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the association of ligand-receptor complexes, experimentally proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissociation. Notably, the rate constants measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand-receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomolecular recognition, intrinsically disordered proteins, and biosensor technology.

Disentangling the recognition complexity of a protein hub using a nanopore.

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiological and disease-like conditions, their reversible interactions with other proteins remain modestly examined. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 derivative and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.

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.

Insertion state of modular protein nanopores into a membrane.

In the past decade, significant progress has been made in the development of new protein nanopores. Despite these advancements, there is a pressing need for the creation of nanopores equipped with relatively large functional groups for the sampling of biomolecular events on their extramembranous side. Here, we designed, produced, and analyzed protein nanopores encompassing a robust truncation of a monomeric ß-barrel membrane protein. An exogenous stably folded protein was anchored within the aqueous phase via a flexible peptide tether of varying length. We have extensively examined the pore-forming properties of these modular protein nanopores using protein engineering and single-molecule electrophysiology. This study revealed distinctions in the nanopore conductance and current fluctuations that arose from tethering the exogenous protein to either the N terminus or the C terminus. Remarkably, these nanopores insert into a planar lipid membrane with one specific conductance among a set of three substate conductance values. Moreover, we demonstrate that the occurrence probabilities of these insertion substates depend on the length of the peptide tether, the orientation of the exogenous protein with respect to the nanopore opening, and the molecular mass of tethered protein. In addition, the three conductance values remain unaltered by major changes in the composition of modular nanopores. The outcomes of this work serve as a platform for further developments in areas of protein engineering of transmembrane pores and biosensor technology.

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.

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy.

This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd , the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd , the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.

Single-Molecule Protein Detection in a Biofluid Using a Quantitative Nanopore Sensor.

Protein detection in complex biological fluids has wide-ranging significance across proteomics and molecular medicine. Existing detectors cannot readily distinguish between specific and nonspecific interactions in a heterogeneous solution. Here, we show that this daunting shortcoming can be overcome by using a protein bait-containing biological nanopore in mammalian serum. The capture and release events of a protein analyte by the tethered protein bait occur outside the nanopore and are accompanied by uniform current openings. Conversely, nonspecific pore penetrations by nontarget components of serum, which take place inside the nanopore, are featured by irregular current blockades. As a result of this unique peculiarity of the readout between specific protein captures and nonspecific pore penetration events, our selective sensor can quantitatively sample proteins at single-molecule precision in a manner distinctive from those employed by prevailing methods. Because our sensor can be integrated into nanofluidic devices and coupled with high-throughput technologies, our approach will have a transformative impact in protein identification and quantification in clinical isolates for disease prognostics and diagnostics.

Interactions of a Polypeptide with a Protein Nanopore Under Crowding Conditions.

Molecular crowding, a ubiquitous feature of the cellular environment, has significant implications in the kinetics and equilibrium of biopolymer interactions. In this study, a single charged polypeptide is exposed to competing forces that drive it into a transmembrane protein pore versus forces that pull it outside. Using single-molecule electrophysiology, we provide compelling experimental evidence that the kinetic details of the polypeptide-pore interactions are substantially affected by high concentrations of less-penetrating polyethylene glycols (PEGs). At a polymer concentration above a critical value, the presence of these neutral macromolecular crowders increases the rate constant of association but decreases the rate constant of dissociation, resulting in a stronger polypeptide-pore interaction. Moreover, a larger-molecular weight PEG exhibits a lower rate constant of association but a higher rate constant of dissociation than those values corresponding to a smaller-molecular weight PEG. These outcomes are in accord with a lower diffusion constant of the polypeptide and higher depletion-attraction forces between the polypeptide and transmembrane protein pore under crowding and confinement conditions.

Real-time measurement of protein-protein interactions at single-molecule resolution using a biological nanopore.

Protein-protein interactions (PPIs) are essential for many cellular processes. However, transient PPIs are difficult to measure at high throughput or in complex biological fluids using existing methods. We engineered a genetically encoded sensor for real-time sampling of transient PPIs at single-molecule resolution. Our sensor comprises a truncated outer membrane protein pore, a flexible tether, a protein receptor and a peptide adaptor. When a protein ligand present in solution binds to the receptor, reversible capture and release events of the receptor can be measured as current transitions between two open substates of the pore. Notably, the binding and release of the receptor by a protein ligand can be unambiguously discriminated in a complex sample containing fetal bovine serum. Our selective nanopore sensor could be applied for single-molecule protein detection, could form the basis for a nanoproteomics platform or might be adapted to build tools for protein profiling and biomarker discovery.

Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics.

Interactions of a membrane protein with a detergent micelle represent a fundamental process with practical implications in structural and chemical biology. Quantitative assessment of the kinetics of protein-detergent complex (PDC) interactions has always been challenged by complicated behavior of both membrane proteins and solubilizing detergents in aqueous phase. Here, we show the kinetic reads of the desorption of maltoside-containing detergents from ß-barrel membrane proteins. Using steady-state fluorescence polarization (FP) anisotropy measurements, we recorded real-time, specific signatures of the PDC interactions. The results of these measurements were used to infer the model-dependent rate constants of association and dissociation of the proteomicelles. Remarkably, the kinetics of the PDC interactions depend on the overall protein charge despite the nonionic nature of the detergent monomers. In the future, this approach might be employed for high-throughput screening of kinetic fingerprints of different membrane proteins stabilized in micelles that contain mixtures of various detergents.

Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases.

Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this process remains ambiguous because the signal-to-noise ratio of most spectroscopic and calorimetric techniques is drastically declined by the presence of protein aggregates in solution. We show that by using steady-state fluorescence polarization (FP) spectroscopy the dissociation of the protein-detergent complex (PDC) can be inspected in real time at detergent concentrations below the critical micelle concentration. This article provides experimental evidence of the coexistence of two distinct phases of the dissociations of detergent monomers from membrane proteins. We first noted a slow detergent predesolvation process, which was accompanied by a relatively modest change in the FP anisotropy, suggesting a small number of dissociated detergent monomers from the proteomicelles. This predesolvation phase was followed by a fast detergent desolvation process, which was highlighted by a major alteration in the FP anisotropy. The durations and rates of these phases were dependent on both the detergent concentration and the interfacial PDC interactions. Further development of this approach might lead to the creation of a new semiquantitative method for the assessment of the kinetics of association and dissociation of proteomicelles.

Quantification of Membrane Protein-Detergent Complex Interactions.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.