The nucleation event of amyloid fibrils is one of the most crucial processes that dictate the timing and rate of the pathology of diseases; however, information regarding how protein molecules associate to produce fibril nuclei is currently limited. In order to explore this issue in more detail, we performed time-resolved small angle X-ray scattering (SAXS) measurements on insulin fibrillation, in combination with additional multidirectional analyses of thioflavin T fluorescence, FTIR spectroscopy, light scattering and light transmittance, during the fibrillation process of bovine insulin. SAXS monitoring revealed that insulin molecules associated into rod-like prefibrillar aggregates in the very early stage of the reaction. After the formation of these early aggregates, they appeared to further coalesce mutually to form larger clusters and the SAXS profiles subsequently showed the further time evolution of conformational development towards mature amyloid fibrils. Distinct types of structural units in terms of shape in a nano-scale order, cross-β content and thioflavin T fluorescence intensity were observed in a manner that was dependent on the fibrillation pathways. These results suggest the presence of diverse substructures that characterize various fibrillation pathways and eventually, manifest polymorphisms in mature amyloid fibrils.

The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two C_H2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of C_H2 with respect to C_H3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of C_H2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.

α-Crystallin possesses a dynamic quaternary structure mediated by its subunit dynamics. Elucidation of a mechanism of subunit dynamics in homo-oligomers of αB-crystallin was tackled through deuteration-assisted small-angle neutron scattering (DA-SANS) and electrospray ionization (ESI) native mass spectrometry (nMS). The existence of subunit exchange was confirmed with DA-SANS, and monomers liberated from the oligomers were observed with nMS. With increasing temperature, an increase in both the exchange rate and monomer population was observed despite the absence of oligomer collapse. It is proposed that transiently liberated subunits, namely, “traveling subunits,” play a role in subunit exchange. Moreover, we propose that protein function is regulated by these traveling subunits.

To understand a biological system, it is important to observe structures of biomolecules in the solution where the system is functionalized. Small-Angle X-ray Scattering coupled with Size Exclusion Chromatography (SEC-SAXS) is one of techniques to selectively observe the target molecules in the multi-component system. However, this technique is believed to be available only with a synchrotron-based SAXS instrument due to requirement of high beam intensity and, therefore, the limitation of the beam time was obstacle to satisfy demands from many bio-researchers. We newly developed Laboratory-based Size exclusion chromatography SAXS System (La-SSS) by utilizing a latest laboratory-based SAXS instrument and finely optimization of the balance between flow rate, cell volume, irradiation time and so on. La-SSS succeeded not only decoupling of target protein(s) from non-specific aggregates but also measurement of each concerned component in a multi-component system. In addition, an option: “stopping mode”, which is designed for improving statistics of SAXS profile, realized a high S/N data acquisition for the most interesting protein in a multi-component system. Furthermore, by utilizing a column having small bed volume, the small-scale SEC-SAXS study makes available. Through optimization of instrumental parameters and environments, La-SSS is highly applicable for experimental requirements from various biological samples. It is strongly expected that a La-SSS concept must be a normal option for laboratory-based SAXS in the near future.

AlphaB crystallin (αB-crystallin) is a key protein for maintaining the long-term transparency of the eye lens. In the eye lens, αB-crystallin is a “dynamical” oligomer regulated by subunit exchange between the oligomers. To elucidate the unsettled mechanism of subunit exchange in αB-crystallin oligomers, the study was carried out at two different protein concentrations, 28.5 mg/mL (dense sample) and 0.45 mg/mL (dilute sample), through inverse contrast matching small-angle neutron scattering. Interestingly, the exchange rate of the dense sample was the same as that of the dilute sample. From analytical ultracentrifuge measurements, the coexistence of small molecular weight components and oligomers was detected, regardless of the protein concentration. The model proposed that subunit exchange could proceed through the assistance of monomers and other small oligomers; the key mechanism is attaching/detaching monomers and other small oligomers to/from oligomers. Moreover, this model successfully reproduced the experimental results for both dense and dilute solutions. It is concluded that the monomer and other small oligomers attaching/detaching mainly regulates the subunit exchange in αB-crystallin oligomer.

In this study, we investigate the α process of a polystyrene thin film using inelastic neutron scattering (INS), dielectric relaxation spectroscopy (DRS), and thermal expansion spectroscopy (TES). The DRS and TES measurements exhibited a decrease in glass transition temperature ($T_{\mathrm{g}}$) with film thickness. On the other hand, an increase in $T_{\mathrm{g}}$ was observed in INS studies. In order to interpret this contradiction, we investigated the temperature dependence of the peak frequency ($f_{\mathrm{m}}$) of the α process probed by DRS and TES. The experiments revealed an increase in the peak frequency ($f_{\mathrm{m}}$) with decreasing film thickness in the frequency region. This observation is consistent with the observed decrease in $T_{\mathrm{g}}$ with thickness. Interestingly, the increase in $T_{\mathrm{g}}$ with film thickness was confirmed by fitting the temperature dependence measurements of the peak frequency with the Vogel-Fulcher-Tammann equation, within the frequency region probed by INS. The discrepancy between INS and DRS or TES descriptions of the α process is likely to be attributed to a decrease in the apparent activation energy with film thickness and reduced mobility, due to the impenetrable wall effect.