Application of cysteinyl prolyl ester for the synthesis of cyclic peptides containing an RGD sequence and their biological activity measurement.

Cysteinyl RGD-peptidyl cysteinyl prolyl esters, which have different configurations at the cysteine and proline residues, were synthesized by the solid-phase method and cyclized by the native chemical ligation reaction. Cyclization efficiently proceeded to give cyclic peptides, regardless of the difference in the configuration. The peptides were further derivatized to the corresponding desulfurized or methylated cyclic peptides at the Cys residues. The inhibition activity to αvß6 integrin binding was then analyzed by ELISA. The results showed that the activity varied depending on the difference in the configuration and modification of the cysteinyl prolyl ester (CPC) moiety, demonstrating the usefulness of this method in the search for a good inhibitor of the protein-protein interaction.

Chemical synthesis of per-selenocysteine human epidermal growth factor.

Human seleno-epidermal growth factor (seleno-EGF), a 53-residue peptide where all six cysteine residues of the parent human EGF sequence were replaced by selenocysteines, was synthesized and the oxidative folding of a polypeptide containing three diselenide bonds was compared to that of the parent cysteine peptide. The crude high performance liquid chromatography (HPLC) profiles clearly showed that both the native EGF and its selenocysteine-analogue fold smoothly, yielding a single sharp peak, proving that even in the case of three disulfide-bonded polypeptides the disulfide-to-diselenide bond substitution is highly isomorphous, as confirmed by conformational circular dichroism measurements and particularly by the biological assays.

Prospective Clinical Evaluation of the Diagnostic Accuracy of a Highly Sensitive Rapid Antigen Test Using Silver Amplification Technology for Emerging SARS-CoV-2 Variants.

The COVID-19 pandemic caused by SARS-CoV-2 remains a serious health concern worldwide due to outbreaks of SARS-CoV-2 variants that can escape vaccine-acquired immunity and infect and transmit more efficiently. Therefore, an appropriate testing method for COVID-19 is essential for effective infection control and the prevention of local outbreaks. Compared to reverse-transcription polymerase chain reaction (RT-PCR) tests, antigen tests are used for simple point-of-care testing, enabling the identification of viral infections. In this study, we tested the clinical usefulness of the FUJIFILM COVID-19 Ag test, an antigen test based on silver amplification and immunochromatographic technology. The FUJIFILM COVID-19 Ag test was shown to detect a lower viral concentration as compared to other conventional kits without significant performance loss in detecting prevalent SARS-CoV-2 variants. We tested nasopharyngeal and nasal swabs from a single patient during two different epidemic periods dominated by various SARS-CoV-2 variants. We observed that the sensitivity of the FUJIFILM COVID-19 Ag test was 95.7% and 85.7% in nasopharyngeal and nasal swabs, respectively. These results suggest that the FUJIFILM COVID-19 Ag test is highly sensitive and applicable when RT-PCR testing is unavailable. Furthermore, these results indicate that high-frequency testing using nasal swab specimens may be a valuable screening strategy.

Liver-expressed antimicrobial peptide 2 functions independently of growth hormone secretagogue receptor in calorie-restricted mice.

Ghrelin is a gastric-derived peptide that stimulates feeding, blood glucose elevation, body temperature reduction, and growth hormone (GH) secretion. Liver-expressed antimicrobial peptide 2 (LEAP2) is an endogenous antagonist of the ghrelin receptor, also called growth hormone secretagogue receptor (GHSR). We studied the effects of LEAP2 administration on feeding, body weight, glycemia, body temperature, and inflammation-related genes in the liver in C57BL/6 J mice and Ghsr-knockout (Ghsr-KO) mice. We found that a single administration of LEAP2 did not abolish fasting-induced food intake in 24-h fasted C57BL/6 J mice or Ghsr-KO mice. Moreover, continuous LEAP2 administration to mice fed ad libitum for 6 days did not affect feeding, body temperature, plasma ghrelin, or blood glucose. By contrast, continuous LEAP2 administration to calorie-restricted C57BL/6 J mice and Ghsr-KO mice induced body weight loss, hypoglycemia, body temperature reduction, and upregulation of Il-6 and Il-1ß mRNAs in the liver. Our findings suggest that LEAP2 functions independently of GHSR, implying that LEAP2 affects physiology beyond the ghrelin-GHSR system.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Chemical synthesis of ferredoxin with 4 selenocysteine residues using a segment condensation method.

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe-2S] cluster. In this paper, we synthesized Se-Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se-Fd was successfully obtained.

Relationships of drooling with motor symptoms and dopamine transporter imaging in drug-naïve Parkinson's disease.

OBJECTIVES: The aim of the present study was to determine the relationships of drooling with motor symptoms and nigrostriatal neuron loss in drug-naïve patients with Parkinson's disease (PD). We therefore examined the relationships of drooling with motor symptoms and striatal dopamine transporter (DAT) binding measured by [123-Iodine]-fluoropropyl-2beta-carbomethoxy-3beta-(4-iodophenylnortropane) dopamine transporter single-photon emission computed tomography(123I-FP-CIT SPECT). PATIENTS AND METHODS: Thirty-five untreated PD patients (14 men and 21 women with a mean age of 71.9 ±â€¯7.2 years) were included in this study. The patients were divided into a drooler group and non-drooler group. They underwent clinical assessments and 123I-FP-CIT SPECT imaging. Motor symptoms were assessed using Unified Parkinson's Disease Rating Scale (UPDRS). RESULTS: The results showed that UPDRS motor score (p = 0.002) and akinetic-rigid score (p = 0.008) were higher and that striatal DAT availability (p = 0.03) was lower in the drooler group than in the non-drooler group. However, tremor score, age, and duration of PD showed no significant differences between the drooler group and non-drooler group. CONCLUSIONS: Drooling in untreated PD is related to an increase in motor symptoms (especially bradykinesia and axial symptoms) and to reduction of striatal DAT availability.

Relations of clinical symptoms with dopamine transporter imaging in drug-naïve Parkinson's disease.

OBJECTIVES: The aim of the present study was to determine the relations of clinical symptoms with nigrostriatal neuron loss in drug-naïve patients with Parkinson's disease (PD). We examined the severity of motor symptoms and freezing of gait (FOG), falls and overactive bladder (OAB) in PD patients and their relations with striatal dopamine transporter (DAT) binding. PATIENTS AND METHODS: Thirty-two untreated PD patients (14 men and 18 women with a mean age of 71.4 ±â€¯7.2 years) were included in this study. Clinical assessments were performed by using Unified Parkinson's Disease Rating Scale (UPDRS) and overactive bladder symptom score (OABSS), and striatal dopamine transporter (DAT) binding was measured by123I-FP-CIT SPECT. RESULTS: The results showed that striatal DAT availability was significantly lower in the high UPDRS motor score group, high akinetic-rigid score group, FOG group, and OAB group than in the low UPDRS motor score group, low akinetic-rigid score group, non-FOG group, and non-OAB group. However, the results also showed that there was no significant difference in striatal DAT availability between the high tremor score group and low tremor score group or between the faller group and non-faller group. CONCLUSIONS: The severity of bradykinesia and axial symptoms and the existence of FOG and OAB in untreated PD patients are related to a decrease in striatal DAT availability. Severity of tremors and occurrence of falls are not related to a decrease in striatal DAT availability. The mechanisms underlying the clinical symptoms of PD involve not only dopaminergic pathways but also non-dopaminergic pathways.

Volume-based parameters on FDG PET may predict the proliferative potential of soft-tissue sarcomas.

INTRODUCTION: Soft-tissue sarcomas (STS) are rare types of tumors that have variable levels of tumor differentiation. F-18 fluorodeoxyglucose positron emission tomography (FDG PET) has been established as an useful tool for STS patients, and the metabolic tumor volume (MTV) and total lesion glycolysis (TLG) are reported to be useful in various cancers. We compared the diagnostic value of four PET parameters (maximum standardized uptake value [SUVmax], SUVmean, MTV, and TLG) from two acquisition timings for predicting the expression of the pathological marker of cell proliferation Ki-67, based on pathological investigation. MATERIALS AND METHODS: In this retrospective study, we investigated 20 patients (59 ± 19 years old, 18-87 years old) with pathologically confirmed STS who underwent FDG PET before surgical intervention. The patients fasted ≥ 6 h before the intravenous injection of FDG. The whole body was scanned twice; at an early phase (61.5 ± 2.6 min) and at a delayed phase (118.0 ± 2.1 min) post-injection. The SUVmax, SUVmean, MTV, and TLG of the primary lesion were measured with a tumor boundary determined by SUV ≥ 2.0. Ki-67 was measured using MIB-1 immunohistochemistry. We used Pearson's correlation coefficient to analyze the relationships between the PET parameters and Ki-67 expressions. The Kaplan-Meier analysis with the log-rank test was performed to compare overall survival between high-group and low-group at each of the four PET parameters and Ki-67 expression. RESULTS: All four PET parameters at each phase showed significant correlations with Ki-67. Among them, the Pearson's correlation coefficient (r) was largest for TLG (r = 0.76 and 0.77 at the early and delayed phases, respectively), followed by MTV (0.70 and 0.72), SUVmax (r = 0.65 and 0.66), and SUVmean (r = 0.62 and r = 0.64). From early to delayed phases, the SUVmax and SUVmean both increased in all 20 patients, whereas the MTV and TLG increased in 13/20 (65%) and 16/20 (80%) patients, respectively. None of the %increases of the PET parameters were significantly correlated with Ki-67. The overall survival was shorter for high-SUVmax, high-SUVmean, high-TLG, and high-Ki-67 groups than the other groups, although the difference did not reach statistical significance. CONCLUSION: The SUVmax, SUVmean, MTV, and TLG acquired at both 1 and 2 h after injection showed significant correlations with Ki-67. Among them, correlation coefficient with Ki-67 expression was highest for TLG, although the best parameter should be determined in a larger population. The delayed-phase FDG PET was equally useful as that of early-phase to predict tumor aggressiveness in STS.

Synthesis of selenocysteine-containing cyclic peptides via tandem N-to-S acyl migration and intramolecular selenocysteine-mediated native chemical ligation.

Selenocysteine-containing cyclic 8-mer peptides, which were designed to mimic the plausible catalytic tetrad of glutathione peroxidase, were successfully synthesized in one pot via tandem N-to-S acyl migration of N-alkylcysteine (NAC)-containing selenopeptides and intramolecular selenocysteine-mediated native chemical ligation (Sec-NCL) of the generated thioesters.

Relation of overactive bladder with motor symptoms and dopamine transporter imaging in drug-naïve Parkinson's disease.

OBJECTIVES: The aim of the present study was to determine the relation of urinary dysfunction with motor symptoms and nigrostriatal neuron loss in drug-naïve patients with Parkinson's disease (PD). We therefore examined the relation of overactive bladder (OAB) symptoms with motor symptoms and striatal dopamine transporter (DAT) binding measured by [123-Iodine]-fluoropropyl-2beta-carbomethoxy-3beta-(4-iodophenylnortropane) dopamine transporter single-photon emission computed tomography (123I-FP-CIT SPECT). PATIENTS AND METHODS: Thirty-one untreated PD patients (12 men and 19 women with a mean age of 71.2 ±â€¯6.7 years) were included in this study. Patients were evaluated with overactive bladder symptom score (OABSS) and divided into an OAB group and Non-OAB group. They underwent clinical assessments and 123I-FP-CIT SPECT imaging. Motor symptoms were assessed using Unified Parkinson's Disease Rating Scale (UPDRS). RESULTS: The results showed that UPDRS motor score (p = 0.01) and akinetic-rigid score (p = 0.002) were higher and that striatal DAT availability (p = 0.01) was lower in the OAB group than in the Non-OAB group. However, tremor score, age, and duration of PD showed no significant differences between the OAB group and Non-OAB group. CONCLUSIONS: Urinary dysfunction in untreated PD is related with increase in motor symptoms (especially bradykinesia and axial symptoms) and reduction of striatal DAT availability.

Sialyl Tn Unit with TFA-Labile Protection Realizes Efficient Synthesis of Sialyl Glycoprotein.

Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.

One-Pot Four-Segment Ligation Using Seleno- and Thioesters: Synthesis of Superoxide Dismutase.

The synthesis of a peptide selenoester was efficiently carried out by the 9-fluorenylmethoxycarbonyl (Fmoc) method using N-alkylcysteine, at the C-terminus of the peptide, as the N-to-S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N,N-diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver-ion-promoted and silver-ion-free thioester activation methods, a one-pot four-segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino-acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.

Preparation of Selenoinsulin as a Long-Lasting Insulin Analogue.

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7UA ,C7UB ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ1/2 ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.

Pyrrole-Based Macrocyclic Small-Molecule Inhibitors That Target Oocyte Maturation.

Polo-like kinase 1 (PLK1) plays crucial roles in various stages of oocyte maturation. Recently, we reported that the peptidomimetic compound AB103-8, which targets the polo box domain (PBD) of PLK1, affects oocyte meiotic maturation and the resumption of meiosis. However, to overcome the drawbacks of peptidic compounds, we designed and synthesized a series of pyrrole-based small-molecule inhibitors and tested them for their effects on the rates of porcine oocyte maturation. Among them, the macrocyclic compound (E/Z)-3-(2,16-dioxo-19-(4-phenylbutyl)-3,19-diazabicyclo[15.2.1]icosa-1(20),6,17-trien-3-yl)propyl dihydrogen phosphate (4) showed the highest inhibitory activity with enhanced inhibition against embryonic blastocyst formation. Furthermore, the addition of this compound to culture media efficiently blocked the maturation of porcine and mouse oocytes, indicating its ability to penetrate the zona pellucida and cell membrane. We investigated mouse oocytes treated with compound 4, and the resulting impairment of spindle formation confirmed PLK1 inhibition. Finally, molecular modeling studies with PLK1 PBD also confirmed the presence of significant interactions between compound 4 and PLK1 PBD binding pocket residues, including those in the phosphate, tyrosine-rich, and pyrrolidine binding pockets. Collectively, these results suggest that the macrocyclic compound 4 may serve as a promising template for the development of novel contraceptive agents.

Reproducibility and uptake time dependency of volume-based parameters on FDG-PET for lung cancer.

BACKGROUND: Volume-based parameters, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG), on F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) are useful for predicting treatment response in nonsmall cell lung cancer (NSCLC). We aimed to examine intra- and inter-operator reproducibility to measure the MTV and TLG, and to estimate their dependency on the uptake time. METHODS: Fifty NSCLC patients underwent preoperative FDG-PET. After an injection of FDG, the whole body was scanned twice: at the early phase (61.4 ± 2.8 min) and delayed phase (117.7 ± 1.6 min). Two operators independently defined the tumor boundary using three different delineation methods: (1) the absolute SUV threshold method (MTVp and TLGp; p = 2.0, 2.5, 3.0, 3.5), (2) the fixed% SUVmax threshold method (MTVq% and TLGq%; q = 35, 40, 45), and (3) the adaptive region-growing method (MTVARG and TLGARG). Parameters were compared between operators and between phases. RESULTS: Both the intra- and inter-operator reproducibility were high for all parameters using any method (intra-class correlation > 0.99 each). MTV3.0 and MTV3.5 resulted in a significant increase from the early to delayed phase (P < 0.05 for both), whereas MTV2.0 and MTV2.5 neither increased nor decreased (P = n.s.). All of the MTVq% values significantly decreased over time (P < 0.01), whereas MTVARG and TLG with any delineation method increased significantly (P < 0.05). CONCLUSIONS: High reproducibility of MTV and TLG was obtained by all of the methods used. MTV2.0 and MTV2.5 were the least sensitive to uptake time, and may be good alternatives when we compare images acquired with different uptake times, although applying constant uptake time is important for volume measurement.

Performance of whole-body integrated 18F-FDG PET/MR in comparison to PET/CT for evaluation of malignant bone lesions.

UNLABELLED: Because of its higher soft-tissue contrast, whole-body integrated PET/MR offers potential advantages over PET/CT for evaluation of bone lesions. However, unlike PET/CT, PET/MR ignores the contribution of cortical bone in the attenuation map. Thus, the aims of this study were to evaluate the diagnostic performance of whole-body integrated (18)F-FDG PET/MR specifically for bone lesions and to analyze differences in standardized uptake value (SUV) quantification between PET/MR and PET/CT. METHODS: One hundred nineteen patients with (18)F-FDG-avid primary malignancies underwent a single-injection, dual-imaging protocol using (18)F-FDG on a PET/CT scanner and a subsequent PET/MR scan with a T1-weighted volumetric interpolated breath-hold examination (VIBE) Dixon sequence for attenuation correction and an unenhanced coronal T1-weighted turbo spin-echo (TSE) sequence for bone analysis. Three sets of images (CT with PET [from PET/CT; set A], T1-weighted VIBE Dixon with PET [set B], and T1-weighted TSE with PET [both from PET/MR; set C]) were analyzed. Two readers rated every lesion using a 4-point scale for lesion conspicuity on PET, a 4-point scale for anatomic allocation of PET-positive lesions, and a 5-point scale for the nature of every lesion based on its appearance on morphologic imaging and uptake on PET. For all lesions and for representative regions of normal bone, SUV analysis was performed for PET/MR and PET/CT. RESULTS: In total, 98 bone lesions were identified in 33 of 119 patients, and 630 regions of normal bone were analyzed. Visual lesion conspicuity on PET was comparable for PET/CT (mean rating, 2.82 ± 0.45) and PET/MR (2.75 ± 0.51; P = 0.3095). Anatomic delineation and allocation of suggestive lesions was significantly superior with T1-weighted TSE MRI (mean rating, 2.84 ± 0.42) compared with CT (2.57 ± 0.54, P = 0.0001) or T1-weighted VIBE Dixon MRI (2.57 ± 0.54, P = 0.0002). No significant difference in correct classification of malignant bone lesions was found among sets A (85/90), B (84/90), and C (86/90). For bone lesions and regions of normal bone, a highly significant correlation existed between the mean SUVs for PET/MR and PET/CT (R = 0.950 and 0.917, respectively, each P < 0.001). However, substantially lower mean SUVs were found for PET/MR than for PET/CT both for bone lesions (12.4% ± 15.5%) and for regions of normal bone (30.1% ± 27.5%). CONCLUSION: Compared with PET/CT, fully integrated whole-body (18)F-FDG PET/MR is technically and clinically robust for evaluation of bone lesions despite differences in attenuation correction. PET/MR, including diagnostic T1-weighted TSE sequences, was superior to PET/CT for anatomic delineation and allocation of bone lesions. This finding might be of clinical relevance in selected cases--for example, primary bone tumors, early bone marrow infiltration, and tumors with low uptake on PET. Thus, a diagnostic T1-weighted TSE sequence is recommended as a routine protocol for oncologic PET/MR.

Model study using designed selenopeptides on the importance of the catalytic triad for the antioxidative functions of glutathione peroxidase.

Although the catalytic triad of glutathione peroxidase (GPx) has been well recognized, there was little evidence for the relevance of the interactions among the triad amino acid residues, i.e., selenocysteine (U), glutamine (Q), and tryptophan (W), to the GPx antioxidative functions. Using a designed selenopeptide having an amino acid sequence of GQAUAWG, we demonstrate here that U, Q, and W present at the active site can interact with each other to exert the enzymatic activity. The amino acid sequence was chosen on the basis of the Monte Carlo molecular simulation for various selenopeptides in polarizable continuous water using the SAAP force field (SAAP-MC). Measurement of the GPx-like activity for the selenopeptide obtained by solid-phase peptide synthesis revealed that the antioxidant activity is cooperatively enhanced by the presence of Q and W proximate to U, although the activity was low compared to selenocystine (U2). The effect of Q on the activity was more important than that of W. In addition, the fluorescence spectrometry suggested a close contact between U and W. These experimental observations were supported by SAAP-MC simulation as well as by ab initio calculation. The latter further suggested that the interaction mode among the triad changes depending on the intermediate states.