Deuteration and selective labeling of alanine methyl groups of ß2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

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 ß2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

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.

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,ß-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,ß-methylene ATP-bound states. Our NMR analyses revealed that, in the α,ß-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

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.

Rapid identification of ligand-binding sites by using an assignment-free NMR approach.

In this study, we developed an assignment-free approach for rapid identification of ligand-binding sites in target proteins by using NMR. With a sophisticated cell-free stable isotope-labeling procedure that introduces (15)N- or (13)C-labels to specific atoms of target proteins, this approach requires only a single series of ligand titrations with labeled targets. Using titration data, ligand-binding sites in the target protein can be identified without time-consuming assignment procedures. We demonstrated the feasibility of this approach by using structurally well-characterized interactions between mitogen-activated protein (MAP) kinase p38α and its inhibitor 2-amino-3-benzyloxypyridine. Furthermore, we confirmed the recently proposed fatty acid binding to p38α and confirmed the fatty acid-binding site in the MAP kinase insert region.

The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana.

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.

Rapid identification of protein-protein interfaces for the construction of a complex model based on multiple unassigned signals by using time-sharing NMR measurements.

Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development.

Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium.

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5 kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.

Direct determination of the insulin-insulin receptor interface using transferred cross-saturation experiments.

Insulin initiates metabolic control by binding to the insulin receptor (IR) on target cells. Kinetic and mutational analyses have revealed two binding sites on the insulin molecule and the residues that compose them. However, direct determination of the insulin-IR interface is required to distinguish those residues that contribute to receptor binding from those required for structural stability. Here, we successfully characterized one binding site using the nuclear magnetic resonance (NMR) transferred cross-saturation method, which can directly determine the binding interface of a large protein-protein complex. The results showed that this binding site contained three residues that have not been identified previously by mutational analyses. On the basis of the structure of the contact site, we also identified a molecule that can displace insulin from the IR. In addition, we discuss the mode of interaction between insulin and its receptor relative to the NMR analyses.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis.

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca(2+)-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

Monitoring the hydrolysis and transglycosylation activity of alpha-glucosidase from Aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry.

Alpha-glucosidase from Aspergillus niger is an enzyme that catalyzes hydrolysis of alpha-1,4 linkages and transglucosylation to form alpha-1,6 linkages. In this study, an analytical method of oligosaccharides by nuclear magnetic resonance (NMR) was used to provide quantitative estimation of the fractions of each sugar unit and was applied to characterize the alpha-glucosidase reaction. Our data indicated that alpha-glucosidase reacts with the nonreducing end of oligosaccharides to form an alpha-1,6 linkage, and then a sugar unit with two alpha-1,6 linkages is gradually produced. Data from mass spectrometry suggested that the sugar unit with two alpha-1,6 linkages originates mainly from a 3mer and/or 4mer when oligosaccharides are used as substrates.

Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. strain S-2.

The structural and enzymatic characteristics of a cutinase-like enzyme (CLE) from Cryptococcus sp. strain S-2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 A resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long-chain substrates, as well as the preferred specificity of FS cutinase for short-chain substrates. In addition, site-directed mutagenesis was performed to increase the hydrolysis activity on long-chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long-chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity.

A simple stabilization method of reduced albumin in blood and plasma for the reduced/oxidized albumin ratio measurement.

Albumin (Alb) is mixture of reduced and oxidized forms. It is physiologically significant to determine Alb(red)%, which is the proportion of reduced Alb in the sum of Alb. However, reduced Alb in both blood and plasma samples is easily converted to oxidized Alb. Accordingly, the stabilization of Alb in samples is necessary to determine precise Alb(red)% values. Alb stabilization in blood or plasma was achieved by pH control and buffer dilution. At least a 50-fold dilution with 50 mmol/l phosphate buffer (pH 6.0) was required for human plasma. For human blood, a 10-fold dilution with 0.5 mol/l sodium citrate buffer (pH 4.3) was required. To measure Alb(red)%, treated samples were applied to HPLC or LC-ESI-TOFMS. We also developed a "pre-incubation method", to accelerate the oxidative reaction in plasma by heating at 37°C. Alb(red)% values were maintained around the initial value for 48 h after stabilizing human plasma and 72 h after stabilizing human blood. Accelerating the oxidative reaction in plasma produced large differences in the Alb(red)% values between normal and model disease samples. Precise Alb(red)% values were routinely obtained under the stabilization control. Additionally, pre-incubation of the plasma before measurement is useful to enhance the difference between normal and disease samples.

Proteome analysis for rat saliva.

Proteome analysis is a popular method to discover biomarkers for the prevention and diagnosis of diseases. Since saliva is a non-invasively available body fluid, gathering of saliva causes minimal harm to patients. Therefore, detection of proteins for the prevention and diagnosis from the saliva sample may be the preferred method, especially for children and elderly people. However, the abundance of salivary proteins and contaminant proteins from food and mouth bacteria obscure identification of proteins present in the saliva at low concentrations. To address this problem, we developed a shotgun proteomic method using two-dimensional nano-flow LC tandem mass spectrometry. We report here that our method is able to detect proteins quantitatively even in small sample volumes of saliva.

Hepatectomy for hepatocellular carcinoma patients who meet the Milan criteria.

BACKGROUND/AIMS: Although hepatocellular carcinoma patients who meet the Milan criteria are optimal candidates for liver transplantation, most such patients in Japan have been treated without liver transplantation. METHODOLOGY: In this retrospective analysis, the patient selection criteria were (1) admission between 1992 and 2005, (2) fulfillment of the Milan criteria, (3) classification within the Barcelona Clinic Liver Cancer stages A1-A4, and (4) no previous anticancer treatment. RESULTS: Of 451 patients who met the selection criteria, 162 underwent hepatectomy. The proportion of patients who underwent hepatectomy was 58% of 106 with stage A1 and 29% of 345 with stages A2-A4. For patients with stages A2-A4, the survival probability after hepatectomy at 3, 5, and 7 years was 89%, 70%, and 61%, respectively. There were no significant differences in survival time between stages A1 and stages A2-A4 after hepatectomy. Among patients with Child-Pugh scores of 5 and 6 in stages A2-A4, 51% and 29% underwent hepatectomy, respectively. CONCLUSIONS: Hepatectomy may be an appropriate first-line treatment option for patients with stages A2-A4 who meet the Milan criteria, when they have a good hepatic reserve and a long waiting time for liver transplantation.

[Structure and function changes of oxidized human serum albumin: physiological significance of the biomarker and importance of sampling conditions for accurate measurement].

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases; however, little is known regarding the pathological and physiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both structural and functional differences between reduced and oxidized HSA. Using LC-ESITOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA, including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions. In addition, we determined blood and plasma sampling conditions for accurate measurement of the oxidized albumin ratio in plasma using EST-TOFMS screening.

Curculin exhibits sweet-tasting and taste-modifying activities through its distinct molecular surfaces.

Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.

Prognostic factors in patients with gemcitabine-refractory pancreatic cancer.

OBJECTIVE: The purpose of this study was to identify prognostic factors in patients with gemcitabine-refractory pancreatic cancer and to determine criteria for selecting candidates for second-line treatment. METHODS: The records of 74 patients who were treated with gemcitabine (GEM) and followed up until disease progression were reviewed retrospectively. Sixteen clinical variables at the time of disease progression after GEM chemotherapy were chosen for analysis in this study. Univariate and multivariate analyses were conducted to identify prognostic factors associated with survival. RESULTS: At the time of analysis, 71 patients had died because of tumor progression. The overall median survival time was 5.1 months after first-line chemotherapy with GEM was initiated. Median survival time after disease progression was 2.0 months. Three factors, performance status, peritoneal dissemination and C-reactive protein level, were identified as independent prognostic factors in multivariate analysis. Median survival time in the good prognosis group (patients with performance status 0 or 1, no peritoneal dissemination and C-reactive protein <5.0 mg/dl) was 3.4 months. CONCLUSIONS: Performance status, serum level of C-reactive protein and peritoneal dissemination were identified as important prognostic factors in patients with GEM-refractory pancreatic cancer. These factors should be considered in determining the treatment following first-line chemotherapy in patients with advanced pancreatic cancer.

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry.

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.