Biology subjects

Kamikubo, H.

Publications and source records attributed to Kamikubo, H..

5 recordsLinked to original sources

bioRxiv2026

Metastable Dimeric Intermediate Drives Native-like Spidroin Fibrillogenesis, while Dehydration Promotes Cross-β Transition

The exceptional mechanical properties of spider dragline silk originate from hierarchical spidroin self-assembly, yet mimicking this process to generate native-like structures remains challenging. Here, we report a metastable dimeric intermediate as an essential molecular factor for fibrillogenesis. Small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed a compact core stabilized by solvent-excluded "dry interfaces" surrounded by extended random coil regions. Within the core, a subset of the polyalanine blocks is held by side-chain packing rather than by main-chain {beta}-sheets; the majority remain exposed in the surrounding coil regions. Upon lowering the denaturant concentration, these exposed polyalanine segments pair with their counterparts at a fibril end, forming intermolecular {beta}-sheets that act as linkers and drive the stepwise end-to-end polymerization of the dimeric particles. In solution, the resulting fibrils exhibit an ordered {beta}-structure in which the polypeptide chains run parallel to the fibril axis, in contrast to amyloid-like cross-{beta}, in which the strands lie perpendicular to it; this matches key features of native silk nanofibril architecture. However, X-ray diffraction (XRD) analysis demonstrates that these native-like nanofibrils undergo an effectively irreversible transition toward amyloid-like cross-{beta} structures upon drying. Native-like fibrils are thus achievable by self-assembly, but structural control is lost during the final dehydration step. Accordingly, we propose a general principle for decoupling self-assembly from dehydration: native-like nanofibrils are formed first, then dried under conditions that restrict molecular-chain mobility (for example, mechanical constraint or immobilization on a surface), which we expect to suppress thermodynamic relaxation into cross-{beta} structures.

biophysics↗

bioRxiv2026

Assembly-pathway regulation dictates pH-responsive actuation in the R-body protein machinery

Refractile bodies (R-bodies) are protein assemblies that form tightly rolled morphologies in cells and undergo large-scale extension into long spirals in response to environmental stimuli. The type 51 R-body, the focus of this study, is assembled from four Reb proteins and undergoes a rapid repeatable [~]50-fold extension. Although R-bodies were first described more than 70 years ago, it has remained unclear how RebC and RebD contribute to the formation and function of this four-protein machinery even though RebA and RebB have been proposed as its major components. We characterized the wild-type R-body (Rb_WT) and a series of reb gene knockout mutants by combining in-cell, biochemical analyses, small-angle X-ray scattering (SAXS) and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. We found that RebD is incorporated into Rb_WT as a minor component, while RebC is not detectably incorporated into the final assembly. Mutants lacking either RebA or RebB still form roll-like assemblies but lack pH-dependent extension. In contrast, mutants lacking RebC and/or RebD exhibit a substantially lower propensity for roll formation and instead undergo off-pathway aggregation with increased {beta}-sheet content. SAXS analyses further indicate that roll-like morphology does not ensure formation of the ordered lamellar architecture characteristic of functional Rb_WT. Thus, pH-responsive R-body actuation is dictated not simply by the major proteins that constitute the final architecture, but by a regulated assembly pathway that builds the ordered lamellar architecture required for actuation. These findings establish assembly-pathway regulation as a key principle for constructing dynamic protein architectures capable of stimuli-responsive mechanical actuation.

biophysics↗

bioRxiv2024

Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1

The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPAR{gamma} is mediated by Transportin-1, but PPAR{gamma} lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPAR{gamma} in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPAR{gamma}, indicating that PPAR{gamma} recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPAR{gamma}.

molecular biology↗

bioRxiv2022

Elucidating polymorphs of crystal structures with intensity-based hierarchical clustering analysis on multiple diffraction datasets

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference for defining a threshold used for grouping the isomorphous datasets ( isomorphic threshold) poses a challenge. Here, we have applied unit cell-based and intensity-based HCAs to the datasets of apo-trypsin and inhibitor-bound trypsin that were mixed post-data acquisition to investigate how effective HCA is in classifying polymorphous datasets. Single-step intensity-based HCA successfully classified polymorphs with a certain isomorphic threshold. In datasets of several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested isomorphic threshold. Polymorphs were also detected in single crystals using the data collected by the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis. SynopsisSingle-step intensity-based hierarchical clustering is demonstrated to allow the detection of structural polymorphs in the diffraction datasets obtained from multiple crystals. By splitting the datasets collected by continuous helical scheme into several chunks, both inter and intra-crystal polymorphs can be successfully analyzed.

molecular biology↗

bioRxiv2021

Interaction of a photochromic UV sensor protein Rc-PYP with PYP-binding protein

Photoactive yellow protein (PYP) from Halorhodospira halophila is one of typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were studied. It was found that UV light-induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-II). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-I) is formed transiently, which produced Complex-II via a second-order reaction. Any other intermediates, including those from pBL do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-II dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor, of which signaling process is similar to that of other light sensor proteins in the visible light region. The photochromic heterogeneous intermolecular interactions formed between PYP and PBP can be used as a novel and useful tool in optogenetics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/446672v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@a62293org.highwire.dtl.DTLVardef@127f8d2org.highwire.dtl.DTLVardef@140b1f2org.highwire.dtl.DTLVardef@629d0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗