Masaaki Sugiyama

Multi-domain proteins (MDPs) adopt diverse conformations arising from cooperative inter-domain motions, and such dynamics are coupled to their biological functions. Quantitative characterization of these motions is crucial for elucidating their functional mechanisms. Although small-angle X-ray scattering (SAXS) provides information on overall domain arrangement, the limited experimental constraints hinder reliable discrimination of conformational ensembles derived from molecular dynamics (MD) simulations. To address this limitation, complementary experimental constraints that offer domain-selective structural information are required. Inverse contrast-matching small-angle neutron scattering (iCM-SANS), combined with segmental deuteration, enables selective observation of individual domains and provides such complementary constraints. However, practical strategies for preparing segmentally deuterated MDPs have yet to be established. Here, we develop an experimental protocol that integrates controlled protein deuteration with high-efficiency multi-step protein ligation to generate a segmentally deuterated MDP. The combined use of SAXS and iCM-SANS yields complementary structural constraints that enhance discrimination of MD-derived conformational ensembles. This protocol expands the applicability of domain-selective observation, enabling analysis of dynamics in MDPs.

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. This multi-technique approach extends previous static structural studies by enabling a parallel comparison of solution-state dynamics among multiple glycosyltransferases. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open–closed interdomain motion and a tendency toward more compact conformations in the presence of substrate, suggesting dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

Cyanobacterial circadian oscillations arise from a phosphorylation cycle of KaiC that is coupled to reversible complex formation with KaiA and KaiB. Although representative assemblies such as A ₂ C ₆ , B ₆ C ₆ , and A ₁₂ B ₆ C ₆ have been structurally characterized, quantitative understanding of their stoichiometry, affinity, and formation kinetics during the oscillation cycle remains limited. Here, we systematically quantified AC, BC and ABC complex formation using phosphorylation-mimetic KaiC and controlled protein mixing ratios by integrating analytical ultracentrifugation with small-angle X-ray/neutron scattering. A ₂ C ₆ formation exhibits graded dependence on the phosphorylation state of KaiC and occurs rapidly in solution. In contrast, B ₆ C ₆ formation behaves in a switch-like manner, showing strong selectivity for the hyperphosphorylation mimic and proceeding on a slow timescale (∼ 6 h). Upon B ₆ C ₆ formation, KaiA rapidly associates with the complex to generate A _n B ₆ C ₆ , with KaiA occupancy n determined by the mixing ratio through fast redistribution among coexisting complexes. This behavior enables dynamic allocation of KaiA among clock complexes. Together, these quantitative insights delineate a hierarchy of assembly dynamics—fast, graded AC exchange; slow, state-selective BC formation; and rapid KaiA redistribution—revealing a mechanistic basis for the dynamic regulation of the Kai oscillator.

Isolating native monomers of oligomeric proteins offers a promising yet underexplored approach to elucidating their functions and identifying drugs that target oligomerization interfaces. Here, we present a strategy for isolating monomers from oligomeric proteins by confinement within spherical Pd(II)-coordination cages. Upon encapsulation of a stable dimeric protein, superoxide dismutase 1 (SOD1), a well-defined cavity of the metallo-cage selectively captured its native monomer. This size-selective isolation enabled direct analysis of monomeric properties, particularly interfacial ligand binding. Saturation transfer difference (STD) NMR revealed that flavonoids, such as quercetin, bind specifically to the encapsulated SOD1 monomer but not to the dimer, indicating interactions at the exposed dimer interface. These findings establish monomer isolation within coordination cages as a strong platform for studying oligomeric proteins.

新試験研究炉を意義あるものにするためには，まずその根本的な理念を明確にし，何を目的とするかを定める必要がある。そして，その実現に向けてどのような活動を行うべきかが重要である。実際に施設が稼働するまでには長い時間がかかるため（10年以上と想定されている），その間にどのような活動を行い，当初の目標を実現するかを考えることが求められる。本論では，新試験研究炉実現に向けて現時点での基本的な考えをできる限り記載する。

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. In the absence of stress, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, large sHsp oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHsps exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHsps from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHsps from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified the specific amino acid residues responsible for the temperature dependency observed. The C-terminal region containing the IXI/V motif and the α-crystallin domain were found to be the primary determinants of oligomer stability and its temperature dependence.

Small heat shock proteins (sHSPs) are ubiquitous, low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. Under normal conditions, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, the large sHSP oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHSPs exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHSPs from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHSPs from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified specific amino acid residues responsible for the observed temperature dependency.

Biomacromolecular solutions inevitably contain impurities in addition to the target biomacromolecules. This has been a major obstacle to achieving high-precision solution scattering measurements. To overcome this problem, small-angle X-ray scattering (SAXS) coupled with size-exclusion chromatography (SEC-SAXS) has been developed. This method involves injecting the solution eluted by SEC directly into a measurement cell and conducting SAXS measurements during the elution of the target biomacromolecule. This technique has resulted in a paradigm shift in biomacromolecule solution scattering. Currently, the application of the SEC–SAXS system to small-angle neutron scattering (SANS) is being advanced. However, since the target biomacromolecules in the sample solution are not only purified but also diluted by SEC and pass through the sample cell in a short time, this method is being implemented in SANS instruments at high neutron flux. Here, we developed a new type of SEC–SANS system that can operate effectively with a SANS instrument at medium neutron flux. Its key innovation is the design and optimization of a dedicated flow path that allows for the storage of only the target biomolecules eluted from SEC in the sample cell. This innovation enables long-duration measurements, termed the `stopping mode', for SEC samples. Consequently, this method allows for acquiring high-precision solution scattering data for target biomacromolecules, enabling SEC–SANS measurements even with SANS instruments at medium neutron flux.

Encapsulation of a single protein within a confined space can lead to distinct properties compared to bulk solutions, but controlling the number of encapsulated proteins and their environment remains challenging. This study demonstrates the encapsulation of single proteins within well‐defined, tunable cavities of self‐assembled coordination cages, thereby enhancing protein stability. Within uniform cavities of size‐tunable coordination cages, 15 different proteins of varying sizes (3–6 nm in diameter) and properties (e.g., isoelectric points and hydrophobicity) were successfully confined. Various analytical techniques confirmed that the proteins maintained their secondary structures and enzymatic activities under denaturing conditions such as exposure to organic solvents, heat, and buffers. These findings suggest that such coordination cages have the potential to serve as synthetic hosts for precisely controlling protein functions within their customizable cavities.