Tohru Terada

Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective 13C-15N labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr35 and Ser39 in the ‘effector region’ (residues 32–40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RBD) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[13C′]proline and L-[15N]threonine, we obtained about 6 mg of Ras protein. As the Pro–Thr sequence is unique in the Ras protein, the Thr35 cross peak of the Ras•Raf RBD complex was unambiguously identified by the 2D 1H–15N HNCO experiment. The Ser39 cross peak was similarly identified with the [13C′]Asp/[15N]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNATyr_CUA (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNATyr_CUA transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of l-[15N]tyrosine. In the gene encoding the Ras protein, the codon for Tyr32 was changed to an amber codon (TAG). This template DNA and the [15N]Tyr-tRNATyr_CUA were reacted for 30 min in 30 mL of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [15N]Tyr32-Ras protein. In the 1H–15N HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr32.

Crystal Structure of the Terminal Oxygenase Component of Cumene Dioxygenase from Pseudomonas fluorescens IP01

The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.

ROKU: a novel method for identification of tissue-specific genes

BackgroundOne of the important goals of microarray research is the identification of genes whose expression is considerably higher or lower in some tissues than in others. We would like to have ways of identifying such tissue-specific genes.ResultsWe describe a method, ROKU, which selects tissue-specific patterns from gene expression data for many tissues and thousands of genes. ROKU ranks genes according to their overall tissue specificity using Shannon entropy and detects tissues specific to each gene if any exist using an outlier detection method. We evaluated the capacity for the detection of various specific expression patterns using synthetic and real data. We observed that ROKU was superior to a conventional entropy-based method in its ability to rank genes according to overall tissue specificity and to detect genes whose expression pattern are specific only to objective tissues.ConclusionROKU is useful for the detection of various tissue-specific expression patterns. The framework is also directly applicable to the selection of diagnostic markers for molecular classification of multiple classes.

Folding free‐energy landscape of a 10‐residue mini‐protein, chignolin

Chignolin is an artificial mini‐protein composed of 10 residues (GYDPETGTWG) that has been shown to cooperatively fold into a β‐hairpin structure in water. We extensively explored the conformational space of chignolin using a 180‐ns multicanonical molecular dynamics (MD) simulation and analyzed its folding free‐energy landscape. In the MD trajectory, we found structures that satisfy 99% of the experimental restraints and are quite close to the experimentally determined structures with Cα root‐mean‐square‐deviations of less than 0.5 Å. These structures formed a large cluster in the conformational space with the largest probability of existence, agreeing well with the experiment.

Minimum free energy path of ligand-induced transition in adenylate kinase.

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme.

An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types

SummaryA combination of calculation and experiment is used to demonstrate that the global fold of larger proteins can be rapidly determined using limited NMR data. The approach involves a combination of heteronuclear triple resonance NMR experiments with protonation of selected residue types in an otherwise completely deuterated protein. This method of labelling produces proteins with α-specific deuteration in the protonated residues, and the results suggest that this will improve the sensitivity of experiments involving correlation of side-chain (1H and 13C) and backbone (1H and 15N) amide resonances. It will allow the rapid assignment of backbone resonances with high sensitivity and the determination of a reasonable structural model of a protein based on limited NOE restraints, an application that is of increasing importance as data from the large number of genome sequencing projects accumulates. The method that we propose should also be of utility in extending the use of NMR spectroscopy to determine the structures of larger proteins.

A method for evaluating multicanonical potential function without iterative refinement: Application to conformational sampling of a globular protein in water

Multicanonical molecular dynamics (MD) is a powerful technique for sampling conformations in rugged potential surfaces, and has been applied to various systems. However, because of the practical difficulty in the iterative refinement of the multicanonical potential function, Emc, a multicanonical MD has not yet been applied to a large system such as a globular protein in explicit solvent. We propose a method to evaluate Emc for a protein in water without the iterative refinement process. The method was applied to the system of a protein, chymotrypsin inhibitor 2 (Ci2), in explicit water, composed of 11 330 atoms. We successfully produced a flat energy distribution covering the energy range corresponding to temperatures from 290 to 400 K. A 10-ns trajectory of the multicanonical MD simulation revealed that Ci2 adopts three different conformations in the long loop (residues 54–63), whereas a 10-ns trajectory of the conventional canonical MD simulation found only one conformation near the initial structure. ...

Human sweet taste receptor mediates acid-induced sweetness of miraculin

Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity.

Acid-induced sweetness of neoculin is ascribed to its pH-dependent agonistic-antagonistic interaction with human sweet taste receptor

Neoculin (NCL) is a sweet protein that also has taste‐modifying activity to convert sourness to sweetness. However, it has been unclear how NCL induces this unique sensation. Here we quantitatively evaluated the pH‐dependent acid‐induced sweetness of NCL using a cell‐based assay system. The human sweet taste receptor, hT1R2‐hT1R3, was functionally expressed in HEK293T cells together with Gα protein. When NCL was applied to the cells under different pH conditions, it activated hT1R2‐hT1R3 in a pH‐dependent manner as the condition changed from pH 8 to 5. The pH‐response sigmoidal curve resembled the imidazole titration curve, suggesting that His residues were involved in the taste‐modifying activity. We then constructed an NCL variant in which all His residues were replaced with Ala and found that the variant elicited strong sweetness at neutral pH as well as at acidic pH. Since NCL and the variant elicited weak and strong sweetness at the same neutral pH, respectively, we applied different proportions of NCL‐variant mixtures to the cells at this pH. As a result, NCL competitively inhibits the variant‐induced receptor activation. All these data suggest that NCL acts as an hT1R2‐hT1R3 agonist at acidic pH but functionally changes into its antagonist at neutral pH.—Nakajima, K., Morita, Y., Koizumi, A., Asakura, T., Terada, T., Ito, K., Shimizu‐Ibuka, A., Maruyama, J., Kitamoto, K., Misaka, T., Abe, K. Acid‐induced sweetness of neoculin is ascribed to its pH‐dependent agonistic‐antagonistic interaction with human sweet taste receptor. FASEB J. 22, 2323–2330 (2008)

Refinement of comparative models of protein structure by using multicanonical molecular dynamics simulations

Comparative modelling is a powerful method that easily predicts a considerably accurate structure of a protein by using a template structure having a similar amino-acid sequence to the target protein. However, in the region where the amino-acid sequence is different between the target and the template, the predicted structure remains unreliable. In such a case, the model has to be refined. In the present study, we explored the possibility of a molecular dynamics-based method, using the human SAP Src Homology 2 (SH2) domain as the modelling target. The multicanonical method was used to alleviate the multiple-minima problem and the generalised Born/surface area model was used to reduce the computational cost. In addition, position restraints were imposed on the atoms in the reliable regions to avoid unnecessary conformational sampling. We analyzed the conformational distribution of the ligand-recognition loop of the domain and found that the most populated conformational clusters in the ensemble of the model agreed well with one of the two major clusters in the ensemble of the reference simulation starting from the crystal structure. This demonstrates that the current refinement method can significantly improve the accuracy of an unreliable region in a comparative model.