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Modulating Protein-Protein Interactions with Visible-Light Responsive Peptide Backbone Photoswitches
摘要： Life relies on a myriad of carefully orchestrated processes, in which proteins and their direct interplay ultimately determine cellular function and disease. Modulation of these complex cross-talks has recently attracted attention, even as a novel therapeutic strategy. Here, we describe the synthesis and characterization of two visible-light responsive peptide backbone photoswitches based on azobenzene derivatives to exert optical control over protein-protein interactions (PPI). Our novel peptidomimetics undergo fast isomerization and reversibility with low photochemical fatigue under alternatively blue/green-light irradiation cycles. Both bind in the nanomolar rage to the protein of interest. Importantly, our best peptidomimetic displays a clear difference between isomers in its protein-binding capacity and, in turn, in its potential to inhibit enzymatic activity via PPI disruption. In addition, crystal structure determination, docking and MD calculations give a molecular interpretation and open new avenues in the design and synthesis of future photoswitchable PPI modulators.
关键词： protein-protein interactions,photopharmacology,visible-light irradiation,azobenzene,photoswitches
更新于2025-11-21 11:20:42
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Protein-Protein Interactions of Highly Concentrated Monoclonal Antibody Solutions via Static Light Scattering and Influence on the Viscosity
摘要： The ability to design and formulate mAbs to minimize attractive interactions at high concentrations is important for protein processing, stability and administration, particularly in subcutaneous delivery, where high viscosities are often challenging. The strength of protein-protein interactions (PPI) of an IgG1 and IgG4 monoclonal antibody (mAb) from low to high concentration were determined by static light scattering (SLS) and used to understand viscosity data. The PPI were tuned using NaCl and five organic ionic co-solutes. The PPI strength was quantified by the normalized structure factor S(0)/S(0)HS and Kirkwood-Buff integral G22/G22,HS (HS = hard sphere) determined from the SLS data, and also by fits with (1) a spherical Yukawa potential and (2) an interacting hard sphere (IHS) model, which describes attraction in terms of hypothetical oligomers. The IHS model was better able to capture the scattering behavior of the more strongly-interacting systems (mAb and/or co-solute) than the spherical Yukawa potential. For each descriptor of PPI, linear correlations were obtained between the viscosity at high concentration (200 mg/mL) and the interaction strengths evaluated both at low (20 mg/mL) and high concentration (200 mg/mL) for a given mAb. However, the only parameter that provided a correlation across both mAbs was the oligomer mass ratio (moligomer/mmonomer+dimer) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.
关键词： Protein-protein interactions,static light scattering,co-solutes,monoclonal antibody,viscosity,interacting hard sphere model,Yukawa potential
更新于2025-09-23 15:23:52
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Surface Plasmon Resonance Based Recent Advances in Understanding Plant Development and Related Processes
摘要： Since its introduction in the early 1990s, SPR has now become a powerful research tool for studying specificity, affinity and real time kinetics of a broad range of biomolecular interactions, including protein-DNA, protein-protein, protein-carbohydrate, protein-RNA and protein-lipid interactions. Surface plasmon resonance (SPR) has provided crucial information on the mechanisms of molecular interactions accompanying varied aspects of plant development. The structure-function relationship of various lectins depends on the quaternary arrangement of its monomers. Novel findings have been made in plant hormone research using SPR as a technique. Thus, new salicylic acid binding proteins (SABPs) have been identified in Arabidopsis. These include α-ketoglutarate dehydrogenase E2 subunit, glutathione S-transferases, the oligopeptidases TOP2 and TOP1, and members of GAPDH protein family. By immobilizing biotin-labelled DELLA peptides on the sensor chip and AtGID1a [Arabidopsis gibberellic acid (GA) receptor] as analyte, GA4 has been observed to maximally enhance binding between DELLA and GID1. Molecularly imprinted monolayer (MIM)-decorated SPR detection method precisely differentiates between similar plant hormones, such as, IAA, 1H-indole-3-butyric acid (IBA) and kinetin (KT), with detection limits around sub-picomolar range. Coronatine Insensitive-1 (COI1) has been shown to act as a jasmonic acid receptor using SPR. Ricin, a plant toxin, was detected at a concentration 2,500 times less than the minimum lethal dose (200 ng.ml-1) using a SPR biosensor. Real time binding kinetic studies of viral proteins (VirE1 and VirE2) and ssDNA using SPR have shown that their binding is strongly influenced by substrate and it occurs at poly T sequences and not at polyA and dsDNA. An interaction between the replicase protein (p93) of cucumber necrosis tombusvirus (CNV) with the host protein, Hsp 70 (molecular chaperone), has revealed the potential role of Hsp90 in the assembly of viral replicase. SPR analysis from a small library of phytochemicals has shown ellagitannin geraniin as one of the most potent inhibitor of Hsp90 (a stabilizer of many oncoproteins). Future applications of SPR technique are likely to provide tremendous inputs into the molecular understanding of plant development and related processes.
关键词： Protein-chaperone interactions,Protein-carbohydrate interactions,Plant viruses,Surface plasmon resonance,Phytohormones,Protein-nucelic acid interactions,Xenobiotics,Protein-protein interactions
更新于2025-09-23 15:21:01
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The Kinetic and Molecular Basis for the Interaction of LexA and Activated RecA Revealed by a Fluorescent Amino Acid Probe
摘要： The bacterial DNA damage response (the SOS response) is a key pathway involved in antibiotic evasion and a promising target for combating acquired antibiotic resistance. Activation of the SOS response is controlled by two proteins: the repressor LexA and the DNA damage sensor RecA. Following DNA damage, direct interaction between RecA and LexA leads to de-repression of the SOS response. However, the exact molecular details of this interaction remain unknown. Here, we employ the fluorescent unnatural amino acid acridonylalanine (Acd) as a minimally-perturbing probe of the E. coli RecA:LexA complex. Using LexA labeled with Acd, we report the first kinetic model for the reversible binding of LexA to activated RecA. We also characterize the effects that specific amino acid truncations or substitutions in LexA have on RecA:LexA binding strength, and demonstrate that a mobile loop encoding LexA residues 75?84 comprises a key recognition interface for RecA. Beyond insights into SOS activation, our approach also further establishes Acd as a sensitive fluorescent probe for investigating the dynamics of protein-protein interactions in other complex systems.
关键词： SOS response,fluorescent amino acid probe,RecA,LexA,protein-protein interactions
更新于2025-09-19 17:13:59
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Probing Specificity of Protein–Protein Interactions with Chiral Plasmonic Nanostructures
摘要： Protein?protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein?protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein?protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody?antigen system. Using surface-immobilized Fab′ fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab′ complexes and random ovalbumin (OVA)-Fab′ aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.
关键词： specificity,Protein?protein interactions,structural order,chiral plasmonic nanostructures,high-throughput screening
更新于2025-09-16 10:30:52
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Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence labelled foci
摘要： In this report we describe the development of a fluorescent protein-protein interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.
关键词： Mdm4,Mdm2,protein-protein interactions,cancer therapy,FLUOPPI,p53
更新于2025-09-11 14:15:04
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Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism
摘要： Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
关键词： coupled folding and binding,synchrotron-radiation circular dichroism,protein-protein interactions,Intrinsically disordered proteins,helix formation
更新于2025-09-11 14:15:04
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Dynamic imaging of small molecule-induced protein-protein interactions in living cells with a fluorophore phase transition-based approach
摘要： Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule-induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule-induced protein-protein association and dissociation in living cells. Here we have applied fluorophore phase transition-based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule-induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drugs (IMiDs)-induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiDs analog (e.g. CC-885)-induced PPI between cereblon and GSPT1. SPPIER can be modified to image small molecule-induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intensive brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.
关键词： GFP,Small molecules,SPPIER,Living cells,Fluorophore phase transition,Protein-protein interactions
更新于2025-09-10 09:29:36
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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
摘要： A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
关键词： Issue 142,cell-cell adhesion,fluorescence correlation spectroscopy,cell-cell interactions,Protein-protein interactions,number and brightness,fluorescence fluctuation spectroscopy,Biochemistry,N&B
更新于2025-09-09 09:28:46
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Hot spots for GPCR signaling: lessons from single-molecule microscopy
摘要： G protein-coupled receptors (GPCRs) are among the best-studied membrane receptors, mainly due to their central role in human physiology, involvement in disease and relevance as drug targets. Although biochemical and pharmacological studies have characterized the main steps in GPCR signaling, how GPCRs produce highly specific responses in our cells remains insufficiently understood. New developments in single-molecule microscopy have made it possible to study the protein–protein interactions at the basis of GPCR signaling in previously inconceivable detail. Using this approach, it was recently possible to follow individual receptors and G proteins as they diffuse, interact and signal on the surface of living cells. This has revealed hot spots on the plasma membrane, where receptors and G proteins undergo transient interactions to produce rapid and local signals. Overall, these recent findings reveal a high degree of dynamicity and complexity in signaling by GPCRs, which provides a new basis to understand how these important receptors produce specific effects and might pave the way to innovative pharmacological approaches.
关键词： single-molecule microscopy,plasma membrane,GPCR signaling,protein–protein interactions,pharmacological approaches
更新于2025-09-04 15:30:14