Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50

Microbial transglutaminase (MTG) has numerous industrial applications in the food and pharmaceutical sectors. Unfortunately, the thermostability of MTG is too low to tolerate the desired conditions used in many of these commercial processes. In a previous study, we used protein engineering to improve the thermostability of MTG. Specifically, we generated a T7C/E58C mutant of MTG from Streptomyces mobaraensis that displayed enhanced resistance to thermal inactivation. In this study, a rational structure-based approach was adopted to introduce a disulfide bridge to further increase the thermostability of MTG. In all, four new mutants, each containing a novel disulfide bond, were engineered. Of these four mutants, D3C/G283C showed the most promising thermostability with a significantly higher ∆T50 (defined as the temperature of incubation at which 50% of the initial activity remains) of + 9 °C by comparison to wild-type MTG. Indeed, D3C/G283C combined enhanced thermostability with a 2.1-fold increased half-life at 65 °C compared with the wild-type enzyme. By structure-based rational design, we were able to create an MTG variant which might be useful for expanding the scope of application in food. • Microbial transglutaminase (MTG) is an enzyme used in many food applications • The applicability of MTG to various industrial processes other than the food sector is being investigated • Improvement of thermostability was confirmed for the disulfide bridge mutant D3C/G283C

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

These authors equally contributed to this work. Conflicts of interest disclosure: Osamu Yokosuka received grant support and honoraria from Bayer. Sadahisa Ogasawara received honoraria from Bayer. The other authors who took part in this study indicated that they did not have anything to declare regarding funding or conflict of interest with respect to this study. Financial support: None. Abstract

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19F-labeling method was 3.5-fold more sensitive than 15N-labeling, and could be combined with other chemical modification techniques such as lysine 13C-methylation. 13C-dimethylated-19F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

Background: Sorafenib has been established as a standard treatment for advanced hepatocellular carcinoma (HCC) for 10 years, however, prognostic factors for patients with advanced HCC receiving sorafenib treatment are presently not well established. Recently, tumor volume represented by Baseline Sum of Longest Diameter (BSLD) of target lesions has been reported as a prognostic factor in some malignancies. The objective of this study was to clarify the prognostic impact of BSLD by Response Evaluation Criteria in Solid Tumor (RECIST) in patients with advanced HCC receiving sorafenib treatment. Methods: We analyzed 232 consecutive patients with advanced HCC who had received sorafenib treatment as first-linechemotherapy from June 2009 to December 2014. Prognostic factors, including the baseline sum longest diameter (BSLD) by RECIST ver1.1 were evaluated by multivariable analysis. Results: Of patients treated with sorafenib (male, 78%; median age, 68 years), 72% had Child-Pugh A and 28% had Child-Pugh B. The median BSLD was 59 mm (range, 10-294). Median time to progression was 3.2 months, and median overall survival (OS) was 9.5 months. By multivariate analysis, BSLD ≥ 60mm (HR 1.55, 95% CI 1.13–2.01, P = 0.02), as well as performance status 1 or 2 (HR 2.01, 95% CI 1.57–2.55, P = 0.01), Child-Pugh class B (HR 2.32, 95% CI 1.78–2.69, P = 0.01), AFP ≥ 400 ng/ml (HR 1.52, 95% CI 1.13–2.01, P = 0.03), and presence of major vascular invasion (HR 1.43, 95% CI 1.08–1.88, P = 0.03) were statistically significant independent predictors of poor prognosis. Median OS of patients with BSLDs ≥ 60 mm and < 60 mm were 5.7 months and 14.5 months, respectively (P = 0.02). Conclusions: BSLD of target lesions by RECIST representing tumor volume is an independent prognostic factor of patients with advanced HCC receiving sorafenib treatment. Legal entity responsible for the study: Chiba University Ethnical Commitee Funding: None Disclosure: T. Chiba: Research grant from Bayer Yakuhin. S. Ogasawara: Receives speaker bureau honoraria. N. Kato: Receives speakers bureau honoraria and research grant from Bayer Yakuhin. All other authors have declared no conflicts of interest.

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for "difficult" proteins, such as membrane proteins and supermolecular complexes.

Significance Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment. In the cases of P2X purinergic receptors, the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. Here, our NMR analyses revealed that the transmembrane region and the membrane side of the lower body exist in conformational equilibrium between the closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s -1 ), and that the small population of the open conformation of zebrafish P2X 4 purinergic receptor causes the partial activation in the α,β-methylene ATP-bound state. These findings provide insights into the mechanism underlying the partial activation of P2X 4 receptors and other ligand-gated ion channels.

Brazzein, a 6.5-kDa protein consisting of 54 amino acids and four disulfide bonds, is the smallest sweet-tasting protein yet isolated from the wild African plant Pentadiplandra brazzeana. Brazzein has various desirable properties for use as a low-calorie sweetener in the diets of individuals suffering from diabetes, obesity, and metabolic syndrome. For example, brazzein has a high water solubility and a high thermostability. In addition, brazzein is 2000-times sweeter than sucrose on a weight basis. Both the solution and crystal structures of brazzein have been reported. In the crystal structure [1], brazzein has a defensin-like fold containing two α-helices and a three-stranded antiparallel β-sheet. Defensins are small cysteine-rich cationic proteins found in both animals and plants, which function by binding to the microbial cell membrane, and, once embedded, forming pore-like membrane defects that allow efflux of essential ions and nutrients. In fact, Yount and Yeaman reported that brazzein has antimicrobial activity against Gram positive (Bacillus subtilis and Staphylococcus aureus) and negative (Escherichia coli) bacteria and a fungus (Candida albicans) at pH 7.5 rather than pH 5.5 [2]. A search for proteins with a similar backbone fold to brazzein using the DALI server shows that structurally similar proteins to brazzein include plant defensins, scorpion neurotoxins (K+ channel blockers), arthropod defensins, mollusc defensins, mold defensins, and a plant trypsin inhibitor. These proteins commonly have a γ-core sequence. Here we compare their sequences, structures and functions, which has led to a conclusion that the C-terminal half of brazzein is important for its antimicrobial activity, brazzein will not have a neurotoxin activity, and it will not act as a trypsin inhibitor.

Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of brazzein reported here will provide additional information and further insights into the intermolecular interaction of brazzein with the sweet-taste receptor.