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Inducible dimerization systems, such as rapamycin‐induced dimerization of FK506 binding protein (FKBP) and FKBP–rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB–FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB–FKBP remains unclear. Here, we report that FRB–FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter‐subunit contacts as in the crystal trimer. These findings expand the applications of the FRB–FKBP oligomerization system in diverse biological events. [ABSTRACT FROM AUTHOR]

SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl‐lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis–Menten analysis and X‐ray crystallography. Kinetic analysis showed that the catalytic efficiency (kcat/Km) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild‐type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction. [ABSTRACT FROM AUTHOR]

Amyloid fibrils of transthyretin (TTR) consist of full‐length TTR and C‐terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C‐terminal fragment remains unclear. Here, we investigated trypsin‐induced aggregation and urea‐induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less‐bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin‐induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin‐induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion. [ABSTRACT FROM AUTHOR]

An acidic environment in bone is essential for bone metabolism and the production of decarboxylated osteocalcin, which functions as a regulatory hormone of glucose metabolism. Here, we describe the high‐resolution X‐ray crystal structure of decarboxylated osteocalcin under acidic conditions. Decarboxylated osteocalcin at pH 2.0 retains the α‐helix structure of native osteocalcin with three γ‐carboxyglutamic acid residues at neutral pH. This implies that decarboxylated osteocalcin is stable under an acidic environment in bone. In addition, site‐directed mutagenesis revealed that Glu17 and Glu21 are important for the adiponectin‐inducing activity of decarboxylated osteocalcin. These findings suggest that the receptor of decarboxylated osteocalcin responds to the negative charge in helix 1 of osteocalcin. [ABSTRACT FROM AUTHOR]

Hydrogen/deuterium (H/D) exchange experiments with cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) are performed at pD 3.4 and 4 degree Celsius to elucidate the structural basis of the initial (D.sub.1) state. It is concluded that the C-terminal [alpha]-helix in the [D.sub.1] state plays an important role in retaining the [D.sub.1] state under the stable conditions and in correctly folding into the native structure of PCP-0SH.

The study employs techniques like NMR and CD calculations to describe the three-dimensional structures of the four tandem repeats of the fibroin-modular-binding protein (FMBP-1), which act as the DNA-binding domain. The analysis shows that the binding of the full-length score and three amino acid peptide repeat leads to an increase in the [alpha]-helicity of the protein regions, which then tend to form a regular secondary structure.

Novel surface plasmon resonance-based biosensor assays for the bioeffect-related screening of chemicals with thyroid-disrupting activity are described. Two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transthyretin (rTTR), were applied in an inhibition assay format in a Biacore 3000 using CM5 biosensor chips coated with L-thyroxine (T4), the main hormone of the thyroid system. Assay conditions were optimized for the natural thyroid hormones, and known thyroid disruptors and structurally related compounds were selected as model compounds to be tested in both assays for their relative potency (RP) compared to T4. The chosen compounds were halogenated phenols, halogenated bisphenols, bisphenol A, 3,5-dichlorobiphenyl, and its hydroxylated metabolite 4-hydroxy-3,5-dichlorobiphenyl (4-OH PCB 14). The TBG-based assay was highly specific for T4, and the rTTR-based assay was sensitive toward several compounds, the highest sensitivity (RP = 4.4) being obtained with 4-OH PCB 14, followed by tetrabromobisphenol A (RP = 1.5) and tetrachlorobisphenol A (RP = 0.75). For the bioeffect-related screening of known and identification of possible new thyroid disruptors, the TPs-based biosensor assays were more sensitive (I[C.sub.50] of 13.7 [+ or -] 1.3 and 8.6 [+ or -] 0.7 nM for the rTTR and the TBG-based assay, respectively), easier to perform, and faster alternatives (10 min/sample) than the currently used methods such as radioligand binding assays and immunoprecipitation-HPLC.

The denaturation process of Natronomonas pharaonis halorhodopsin (NpHR) solubilized with octyl-beta-D-glucopyranoside (OG) was investigated to clarify the effects of the presence of chloride ion and pH on the stability and bleaching of the NpHR chromophore. NpHR and Halobacterium salinarum HR (HsHR) exhibit slight differences in stability in a chloride-free system, as well as in terms of light--dark adaptation and anion selectivity for pumping.

Research has been conducted on equine lysozyme, a calcium-binding lysozyme. The construction of the chimeric protein has been carried out via substitution of the alpha-lactalbumin fluctuating loop with the D-helix of equine lysozyme and the results suggest that this replacement may be useful in stabilizing protein structure.