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Alpha-synuclein (aSyn) is an intrinsically disordered protein (IDP) that can form amyloid fibrils. Fibrils of aSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of aSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of aSyn is sensitive to solution conditions, suggesting that fibril formation of aSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because IDPs such as aSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering (QENS) and small-angle X-ray scattering (SAXS) techniques, we investigated the dynamic and structural properties of aSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The SAXS measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The QENS measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of aSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

Cannabis sativa L. produces unique secondary metabolites known as cannabinoids which have psychotropic and pharmacological activities. Major cannabinoids including DELTA9-tetrahydrocannabinol are biosynthesized from a common precursor olivetolic acid. A type III polyketide synthetase (PKS) from C. sativa named PKS-1 is considered to synthesize the tetraketide intermediate of olivetolic acid by condensation of hexanoyl-CoA with three malonyl-CoA molecules. Whereas most plant PKSs can react with various CoA esters, PKS-1 exhibits a relatively strict substrate specificity for hexanoyl-CoA as the starter substrate. The unique enzymatic property of PKS-1 is important in the cannabinoid pathway. To clarify the structure-function relationship of PKS-1, we have determined X-ray crystal structures of recombinant apo PKS-1 and complexes with hexanoyl-CoA or malonyl-CoA, at resolutions of 1.6 Å, 1.6 Å and 1.8 Å, respectively. The overall structure of PKS-1 is similar to those of other plant PKSs. As for the loop regions around the active site of PKS-1, which are thought to characterize the enzymatic properties, their structures are most similar to those of pyrone synthase from Gerbera hybrida. The binding pattern of the CoA moiety to PKS-1 is similar between hexanoyl-CoA and malonyl-CoA. Compared to other plant PKSs, several amino acid side chains of PKS-1 that constitute the active site cavity are positioned to effectively reduce the cavity volume and bind the aliphatic moiety of hexanoyl-CoA tightly. These structural features of PKS-1 are considered to enable selective binding of hexanoyl-CoA leading to production of cannabinoids unique to C. sativa., 第20回日本蛋白質科学会年会

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.

Hemoglobin (Hb) is a prototypical allosteric protein. We investigated the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that, whereas the deoxy-form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude, but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin, and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.