Yoshihiro Mori

Unfolding and aggregation of transthyretin by the truncation of 50 N-terminal amino acids

Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wild‐type transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C‐terminal fragments starting near amino acid residue 50 as well as full‐length transthyretin. Although a number of previous studies suggest the importance of the C‐terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C‐terminal fragments of transthyretin. To understand the role of C‐terminal fragments in SSA, we examined the effects of the truncation of the N‐terminal portions on the structure and aggregation properties of wild‐type transthyretin. The deletion mutant lacking 50 N‐terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self‐assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N‐terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self‐assembly into aggregations in SSA. Proteins 2008.

The structure of S100A11 fragment explains a local structural change induced by phosphorylation.

S100A11 protein is a member of the S100 family containing two EF‐hand motifs. It undergoes phophorylation on residue T10 after cell stimulation such as an increase in Ca2+ concentration. Phosphorylated S100A11 can be recognized by its target protein, nucleolin. Although S100A11 is initially expressed in the cytoplasm, it is transported to the nucleus by the action of nucleolin. In the nucleus, S100A11 suppresses the growth of keratinocytes through p21CIP1/WAF1 activation and induces cell differentiation. Interestingly, the N‐terminal fragment of S100A11 has the same activity as the full‐length protein; i.e. it is phosphorylated in vivo and binds to nucleolin. In addition, this fragment leads to the arrest of cultured keratinocyte growth. We examined the solution structure of this fragment peptide and explored its structural properties before and after phosphorylation. In a trifluoroethanol solution, the peptide adopts the α‐helical structure just as the corresponding region of the full‐length S100A11. Phosphorylation induces a disruption of the N‐capping conformation of the α‐helix, and has a tendency to perturb its surrounding structure. Therefore, the phosphorylated threonine lies in the N‐terminal edge of the α‐helix. This local structural change can reasonably explain why the phosphorylation of a residue that is initially buried in the interior of protein allows it to be recognized by the binding partner. Copyright

Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.

Effects of the stabilization of the molten globule state on the folding mechanism of α‐lactalbumin: A study of a chimera of bovine and human α‐lactalbumin

The N‐terminal half of the α‐domain (residues 1 to 34) is more important for the stability of the acid‐induced molten globule state of α‐lactalbumin than the C‐terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human α‐lactalbumin and the remainder of the sequence from bovine α‐lactalbumin, were studied by stopped‐flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine α‐lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine α‐lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A‐ and B‐helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1–34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of α‐lactalbumin. Proteins 2005.

(1Z,2Z)-1,2-Bis(3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene)hydrazine

The title compound, C16H14N4S2, crystallizes in symmetry group C2. The molecule is planar with C2h symmetry, with the inversion centre at the mid-point of the hydrazine N-N bond, and it has an N-N s-trans conformation and a Z,Z configuration. The particular crystal examined was a racemic twin, as suggested by the Flack parameter of 0.41 (2) [Flack (1983). Acta Cryst. A39, 876-881].