Label-free biosensors, including conventional quartz-crystal-microbalance (QCM) biosensors, are seriously affected by nonspecific adsorption of contaminants involved in analyte solution, and it is ...exceptionally difficult to extract the sensor responses caused only by the targets. In this study, we reveal that this difficulty can be overcome with an ultrahigh-frequency, wireless QCM biosensor. The sensitivity of a QCM biosensor dramatically improves when the quartz resonator is thinned, which also makes the resonance frequency higher, causing high-speed surface movement. Contaminants weakly (nonspecifically) interact with the quartz surface, but they fail to follow the fast surface movement and cannot be detected as the loaded mass. The targets are, however, tightly captured by the receptor proteins immobilized on the surface, and they can move with the surface, contributing to the loaded mass and decreasing the resonant frequency. We have developed a MEMS QCM biosensor in which an AT-cut quartz resonator, 26 μm thick, is packaged without fixing, and we demonstrate this phenomenon by comparing the frequency changes of the fundamental (∼64 MHz) and ninth (∼576 MHz) modes. At ultrahigh-frequency operation with the ninth mode, the sensor response is independent of the amount of impurity proteins, and the binding affinity is unchanged. We then applied this method to the label-free and sandwich-free, direct detection of C-reactive protein (CRP) in serum and confirmed its applicability.
Time-resolved single-molecule observations by high-speed atomic force microscopy (HS-AFM), have greatly advanced our understanding of how proteins operate to fulfill their unique functions. Using ...this device, we succeeded in visualizing two members of the protein disulfide isomerase family (PDIs) that act to catalyze oxidative folding and reductive unfolding in the endoplasmic reticulum (ER). ERdj5, an ER-resident disulfide reductase that promotes ER-associated degradation, reduces nonnative disulfide bonds of misfolded proteins utilizing the dynamics of its N-terminal and C-terminal clusters. With unfolded substrates, canonical PDI assembles to form a face-to-face dimer with a central hydrophobic cavity and multiple redox-active sites to accelerate oxidative folding inside the cavity. Altogether, PDIs exert highly dynamic mechanisms to ensure the protein quality control in the ER.
Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase ...(PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.
Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these ...combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.
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•We develop an multichannel optimized sonoreactor for amyloid-fibril assays.•The sonoreactor achieves the reproducible amyloid-fibril assays.•The sonoreactor detects ultra-trace 100 ...fM seeds at an accelerated rate.•The primary nucleation and fragmentation pathways are accelerated by ultrasonication.
Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using β2-microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation.
Abstract
A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, ...via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure.
Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. ...However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.
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•Conformational dynamics of the C. thermophilum Hsp104 (CtHsp104) disaggregase is reported•Identification of conformational changes and splitting of the CtHsp104 hexameric ring•The split hexameric ring conformation represents an off-pathway state of CtHsp104
Hsp104 assembles hexameric ring to unfold and regenerate denatured protein aggregates in cells. Inoue et al. identified split conformation of Chaetomium thermophilum Hsp104 disaggregase using X-ray crystallography, cryo-EM, and high-speed atomic force microscopy (HS-AFM) analysis. The split conformation provides insights into an off-pathway state of Hsp104 during disaggregation.
α-Synuclein aggregates, a key hallmark of the pathogenesis of Parkinson's disease, can be amplified by using their seeding activity, and the evaluation of the seeding activity of cerebrospinal fluid ...(CSF) is reportedly useful for diagnosis. However, conventional shaking-based assays are time-consuming procedures, and the clinical significance of the diversity of seeding activity among patients remains to be clarified. Previously, we reported a high-throughput ultrasonication-induced amyloid fibrillation assay. Here, we adapted this assay to amplify and detect α-synuclein aggregates from CSF, and investigated the correlation between seeding activity and clinical indicators. We confirmed that this assay could detect α-synuclein aggregates prepared in vitro and also aggregates released from cultured cells. The seeding activity of CSF correlated with the levels of α-synuclein oligomers measured by an enzyme-linked immunosorbent assay. Moreover, the seeding activity of CSF from patients with Parkinson's disease was higher than that of control patients. Notably, the lag time of patients with Parkinson's disease was significantly correlated with the MIBG heart-to-mediastinum ratio. These findings showed that our ultrasonication-based assay can rapidly amplify misfolded α-synuclein and can evaluate the seeding activity of CSF.
ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that ...enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less-efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.
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•A crystal structure of ERdj5 with a different cluster orientation was solved•HS-AFM visualized rapid motion of the ERdj5 clusters at the single-molecule level•Dynamics of the ERdj5 clusters play an essential role in the ERAD enhancement•Efficient substrate transfer between ERdj5 and BiP requires their dynamic interplay
Maegawa et al. describe a crystal structure of ERdj5, an ER-resident disulfide reductase, with a different cluster orientation from the original structure. High-speed AFM analysis revealed the highly dynamic nature of ERdj5 as a structural feature essential for efficient ERAD of aberrant disulfide-linked protein oligomers.
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