Liviu Movileanu

Interactions between receptor tyrosine kinases and their specific growth factor ligands are crucial for cell signaling. Although much research has focused on these processes, quantitative attention to their earliest kinetics has been limited. Here, we used two immobilization-based sensing methods, biolayer interferometry (BLI) and surface plasmon resonance (SPR), to analyze the real-time binding kinetics of three high-affinity ligands with the full extracellular domain of various epidermal growth factor receptor (EGFR) isoforms. We observed that BLI measurements show both fast and slow dissociation phases, regardless of the EGFR isoform. SPR experiments, despite using a different detection readout, confirm the existence of two binding substates, indicating that this is a common feature of ligand-EGFR interactions. Furthermore, our data systematically demonstrate that the type of immobilization-based sensing technique influences not only the magnitude of kinetic and affinity parameters but also the relative interactions among different ligands and EGFR isoforms. Using these methods, we also found that glycan side chains at position N151 within the canonical ligand-binding site do not affect overall interactions with the three common high-affinity growth factors. Finally, we show that an extensively deglycosylated EGFR isoform binds all tested ligands with significantly lower affinity. Our approach could be applied to other ligand-receptor systems to directly evaluate how specific posttranslational modifications impact their interactions.

Liquid-liquid phase separation (LLPS) is pivotal in generating membraneless organelles and assembling cellular inclusions. Interactions mediated by RNA and intrinsically disordered regions of proteins are ubiquitous mechanisms that drive their LLPS. Here, we identify that a site-specific interaction stimulates the LLPS of WDR5, a chromatin-associated protein hub. Our study proves that WDR5 undergoes self-association between its N-terminal intrinsically disordered region and a multitasking binding site. This mechanism facilitates the formation of liquid droplets in a cell-free environment. Notably, WDR5 undergoes phase separation in mammalian cells, forming nuclear puncta (NP) in response to osmotic stress. Further, nuclear WDR5 condensates encompass a critical oncoprotein transcription factor, MYC, and WDR5-binding RNA under hyperosmotic conditions. Our findings suggest that RNA modulates WDR5 phase separation and influences nuclear puncta formation, potentially serving as a general stress response mechanism. These outcomes illuminate a distinctive mechanochemical signaling process, highlighting the functional interplay among WDR5, RNA, and MYC at the chromatin level, particularly during osmotically induced LLPS.

A ubiquitous problem in protein analytics and medical biotechnology is assessing the interaction of a membrane protein receptor with its cognate protein ligand. This task generally requires transferring the receptor from native membranes or other expression host systems into supported lipid bilayers, liposomes, or nanodiscs. Such a reintegration process necessitates multiple steps for protein solubilization, renaturing, and functional reconstitution. Here, we opportunistically show that biolayer interferometry (BLI) can be directly utilized to evaluate the pre-equilibrium binding kinetics of a membrane protein receptor with its protein ligand in a label-free and membraneless setting. We present real-time measurements probing the association and dissociation phases of these transient complexes, conducted at a high signal-to-noise ratio using free proteomicelles in solution. As a proof-of-concept, we employ a subset of synthetic membrane proteins equipped with a programmable antibody mimetic binder that targets a specific protein ligand. Proteomicelles containing these binder-equipped membrane proteins exhibit high-affinity interactions with ligands attached to the sensor surface. These determinations are further validated by closely related surface plasmon resonance (SPR) measurements of the binder-ligand and proteomicelle-ligand interactions. Finally, this approach is amenable to high-throughput data collection, and its conceptual formulation is potentially extendable to other membrane proteins.

Coordinated interactions between a protein hub, or receptor, and its cognate protein ligands are at the heart of cell signaling. Any significant perturbations in their kinetic and dynamic complexities result in major alterations in biochemical traffic at the subcellular and extracellular levels. The coexistence of multiple ligands with varying local concentrations and affinity constants, as well as the transient nature of their underlying protein-protein interactions (PPIs), makes predicting hub occupancy a challenging task. Here, we develop models of PPIs anchored in queuing theory to determine hub occupancy as a function of the kinetic rate constants and concentrations in complex mixtures of protein ligands. We find that in a ternary mixture of protein ligands spanning a range of kinetic rate constants, the concentration of one ligand can significantly influence the competitive PPIs between the other two ligands and the protein receptor, thereby impacting its overall occupancy. Further, for more complex mixtures, we developed a coarse-graining approach to compartmentalize large numbers of ligands competing for the same binding site of the receptor. Our analytical strategy provides a mechanistic and quantitative understanding of competitive PPIs, with broad applicability to biochemical processes, protein analytics, and drug development.

Two or more protein ligands may compete against each other to interact transiently with a protein receptor. While this is a ubiquitous phenomenon in cell signaling, existing technologies cannot identify its kinetic complexity because specific subpopulations of binding events of different ligands are hidden in the averaging process in an ensemble. In addition, the limited time resolution of prevailing methods makes detecting and discriminating binding events among diverse interacting partners challenging. Here, we utilize a genetically encoded nanopore sensor to disentangle competitive protein-protein interactions (PPIs) in a one-on-one and label-free fashion. Our measurements involve binary mixtures of protein ligands of varying binding affinity against the same receptor, which was externally immobilized on the nanopore tip. We use the resistive-pulse technique to monitor the kinetics and dynamics of reversible PPIs without the nanopore confinement, with a high-time bandwidth, and at titratable ligand concentrations. In this way, we systematically evaluate how individual protein ligands take their turn to reside on the receptor's binding site. Further, our single-molecule determinations of these interactions are quantitatively compared with data generated by a two-ligand, one-receptor queuing model. The outcomes of this work provide a fundamental basis for future developments aimed at a better mechanistic understanding of competitive PPIs. Moreover, they may also form a platform in drug development pipelines targeting high-complexity PPIs mediated by protein hubs.

Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack of cofactor-dependent active sites of transcription factors further complicate the quantitative analysis of these critical processes. Here, we create a genetically encoded label-free sensor to identify the interaction between a motif of the MYC transcription factor, a primary cancer driver, and WDR5, a chromatin-associated protein hub. Using an engineered nanopore equipped with this motif, WDR5 is probed through reversible captures and releases in a one-by-one and time-resolved fashion. Our single-molecule kinetic measurements indicate a weak-affinity interaction arising from a relatively slow complex association and a fast dissociation of WDR5 from the tethered motif. Further, we validate this subtle interaction by determinations in an ensemble using single nanodisc-wrapped nanopores immobilized on a biolayer interferometry sensor. This study also provides the proof-of-concept for a sensor that reveals unique recognition signatures of different protein binding sites. Our foundational work may be further developed to produce sensing elements for analytical proteomics and cancer nanomedicine.

β 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 β 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 β 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 β 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.

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.