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21 results on '"Jun Funahashi"'

1. The Noncompetitive Antagonism of Histamine H1 Receptors Expressed in Chinese Hamster Ovary Cells by Olopatadine Hydrochloride: Its Potency and Molecular Mechanism

Author

Kozue Hayashi, Kiyotoshi Mori, Yuichi Matsumoto, Jun Funahashi, and Hiroshi Yano

Subjects
Pharmacology, medicine.medical_specialty, Chinese hamster ovary cell, chemistry.chemical_element, General Medicine, Histamine H1 receptor, Calcium, Olopatadine Hydrochloride, chemistry.chemical_compound, Histamine receptor, Endocrinology, Histamine H2 receptor, chemistry, Internal medicine, medicine, Antagonism, Histamine
Abstract

Calcium responses to various concentrations of histamine were monitored in Chinese hamster ovary cells stably expressing the human histamine H1 receptor. The effects of various histamine H1 receptor antagonists on the dose-response curve for histamine were evaluated. Olopatadine hydrochloride (olopatadine) inhibited the histamine-induced maximum response (pD2’: 7.5) but had insignificant effects on histamine EC50 values. This noncompetitive property exhibited by olopatadine, which was also observed in human umbilical vein endothelial cells, was the most striking among the antihistamines tested in this study. The geometrical isomer of olopatadine (E-isomer), which had a similar binding affinity to the histamine H1 receptor as olopatadine, showed a mixed antagonistic profile (competitive and noncompetitive). These results indicate that the geometry around the double bond in the dimethylaminopropylidene group is critical for the potent noncompetitive property of olopatadine. Furthermore, binding mode analyses suggest that the protonated amine group in the dimethylaminopropylidene moiety of olopatadine forms an ionic bond with Glu 181 that is present in the second extracellular loop of the histamine H1 receptor, whereas the amine group of the E-isomer does not. The second extracellular loop in aminergic G-protein-coupled receptors contributes to ligand binding and therefore the noncompetitive property of olopatadine may be explained by the interaction with Glu 181.

Published
2008
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2. How can free energy component analysis explain the difference in protein stability caused by amino acid substitutions? Effect of three hydrophobic mutations at the 56th residue on the stability of human lysozyme

Author

Yuji Sugita, Akio Kitao, Katsuhide Yutani, and Jun Funahashi

Subjects
Protein Folding, Mutant, Bioengineering, Biochemistry, Accessible surface area, symbols.namesake, Molecular dynamics, Protein structure, Enzyme Stability, Humans, Computer Simulation, Thermal stability, Molecular Biology, chemistry.chemical_classification, Protein Structure, Tertiary, Amino acid, Gibbs free energy, Crystallography, Amino Acid Substitution, chemistry, Mutation, Biophysics, symbols, Thermodynamics, Muramidase, Protein folding, sense organs, Biotechnology
Abstract

To elucidate the molecular mechanism of thermal stability, it is essential to determine what are the major free energy components that contribute significantly to the total free energy difference caused by amino acid mutations. In this work, we carried out free energy calculations based on all-atom molecular dynamics simulations to investigate the effect of three hydrophobic mutations at the same position, I56A, I56V and I56F of human lysozyme. The calculated free energy differences are in good agreement with the experimental values in all cases. From free energy component analysis, we found that small changes in stability in the I56A and I56V mutants originate from the short-range Lennard-Jones interactions, whereas the I56F mutant is largely destabilized owing to the changes in the long-range electrostatic interactions. The calculated results are also compared with the free energy components determined by an empirical relationship based on the native-state structure and thermodynamic data. Although this relationship has been shown to be very successful in reproducing the stability changes caused by various amino acid substitutions in several proteins, the changes of stability in I56V and I56F mutants are not reproduced very well. By comparing the free energy components calculated by these two approaches, we showed that the effect of the long-range interaction on the stability changes may be underestimated in the empirical relationships when the structural change caused by mutation is relatively small, as in I56F. It is also suggested that estimation of the change in accessible surface area, deltadeltaASA, may be overestimated if the structure around the mutation site in the denatured state is native-like, which would cause overestimation of the free energy change as in the case of I56V. Our results clearly show that the combined approach of the free energy calculation based on the all-atom molecular dynamics simulation and the empirical relationships is very useful for understanding the detailed mechanism of protein stability.

Published
2003
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3. Are the parameters of various stabilization factors estimated from mutant human lysozymes compatible with other proteins?

Author

Katsuhide Yutani, Kazufumi Takano, and Jun Funahashi

Subjects
Protein Folding, Protein Conformation, Mutant, Bioengineering, Biology, Arginine, Crystallography, X-Ray, Biochemistry, Protein Structure, Secondary, chemistry.chemical_compound, Protein stability, Enzyme Stability, Bacteriophage T4, Humans, Molecular Biology, Calorimetry, Differential Scanning, Lysine, Proteins, Amino Acid Substitution, chemistry, Chemical engineering, Mutation, Mutagenesis, Site-Directed, Biophysics, Thermodynamics, Muramidase, Asparagine, Lysozyme, Biotechnology
Abstract

The various factors which contribute to protein stability have been extensively examined using mutant proteins, but the same kinds of substitutions have given different results depending on the substitution sites. Recently, the contributions of some stabilization factors have been quantitatively derived as parameters by a unique equation, considering the conformational changes due to the mutations using mutant human lysozymes [Funahashi et al. (1999) Protein ENG: 12, 841-850]. To evaluate these parameters estimated from the mutant human lysozymes, stability-structure datasets for the mutant T4 lysozymes were selected. The stabilities for the mutant T4 lysozymes could be roughly estimated using these parameters. Notable differences between the estimated and experimental stabilities were caused by the uncertainty in part of the structures due to some Arg and Lys residues fluctuating on the surface of the T4 lysozyme. Excluding these atoms from the estimation gave a good correlation between the estimated and experimental stabilities. These results suggest that the parameters of the various stabilization factors derived from the mutant human lysozymes are compatible with the mutant T4 lysozymes, although they should be improved with respect to some points using more information.

Published
2001
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4. The stability and folding process of amyloidogenic mutant human lysozymes

Author

Jun Funahashi, Katsuhide Yutani, and Kazufumi Takano

Subjects
Folding (chemistry), symbols.namesake, Crystallography, Amyloid, Chemistry, Mutant, Native state, symbols, Mutagenesis (molecular biology technique), Protein folding, Denaturation (biochemistry), Biochemistry, Gibbs free energy
Abstract

Amyloid deposits are frequently formed by mutant proteins that have a lower stability than the wild-type proteins. Some reports, however, have shown that mutant-induced thermodynamic destabilization is not always a general mechanism of amyloid formation. To obtain a better understanding of the mechanism of amyloid fibril formation, we show in this study that equilibrium and kinetic refolding-unfolding reaction experiments with two amyloidogenic mutant human lysozymes (I56T and D67H) yield folding pathways that can be drawn as Gibbs energy diagrams. The equilibrium stabilities between the native and denatured states of both mutant proteins were decreased, but the degrees of instability were different. The Gibbs energy diagrams of the folding process reveal that the Gibbs energy change between the native and folding intermediate states was similar for both proteins, and also that the activation Gibbs energy change from the native state to the transition state decreased. Our results confirm that the tendency to favor the intermediate of denaturation facilitates amyloid formation by the mutant human lysozymes more than equilibrium destabilization between the native and completely denatured states does.

Published
2001
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5. Role of Surface Hydrophobic Residues in the Conformational Stability of Human Lysozyme at Three Different Positions

Author

Yuriko Yamagata, Katsuhide Yutani, Kazufumi Takano, and Jun Funahashi

Subjects
Protein Conformation, Phenylalanine, Mutant, Glycine, Calorimetry, Crystallography, X-Ray, Biochemistry, symbols.namesake, chemistry.chemical_compound, Methionine, Protein structure, Leucine, Enzyme Stability, Hydrolase, Humans, Denaturation (biochemistry), Amino Acids, Isoleucine, Alanine, Calorimetry, Differential Scanning, Hydrogen bond, Chemistry, Membrane Proteins, Valine, Gibbs free energy, Crystallography, Amino Acid Substitution, Mutagenesis, Site-Directed, symbols, Thermodynamics, Muramidase, Lysozyme
Abstract

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes (Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positions on the surface, which are located in the alpha-helix (Val 110), the beta-sheet (Val 2), and the loop (Val 74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by X-ray crystallography at 100 K, respectively. Differences in the denaturation Gibbs energy change between the wild-type and the hydrophobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, and 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identical substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in the stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary structural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutation sites of the exceptional mutant proteins were affected due to the substitutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. These results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.

Published
2000
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6. Contribution of Hydrogen Bonds to the Conformational Stability of Human Lysozyme: Calorimetry and X-ray Analysis of Six Ser → Ala Mutants

Author

Kazufumi Takano, Jun Funahashi, Yuriko Yamagata, Katsuhide Yutani, Masahiro Kubota, and Satoshi Fujii

Subjects
Models, Molecular, Protein Conformation, Calorimetry, Crystallography, X-Ray, Biochemistry, Structure-Activity Relationship, chemistry.chemical_compound, Differential scanning calorimetry, Enzyme Stability, Serine, Humans, Bound water, Molecule, Denaturation (biochemistry), Alanine, Calorimetry, Differential Scanning, Hydrogen bond, Chemistry, Hydrogen Bonding, Crystallography, Energy Transfer, Intramolecular force, Mutagenesis, Site-Directed, Thermodynamics, Muramidase, Lysozyme
Abstract

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.

Published
1999
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7. Contributory presentations/posters

Author

N. Manoj, V. R. Srinivas, A. Surolia, M. Vijayan, K. Suguna, R. Ravishankar, R. Schwarzenbacher, K. Zeth, null Diederichs, G. M. Kostner, A. Gries, P. Laggner, R. Prassl, null Madhusudan, Pearl Akamine, Nguyen-huu Xuong, Susan S. Taylor, M. Bidva Sagar, K. Saikrishnan, S. Roy, K. Purnapatre, P. Handa, U. Varshney, B. K. Biswal, N. Sukumar, J. K. Mohana Rao, A. Johnson, Vasantha Pattabhi, S. Sri Krishna, Mira Sastri, H. S. Savithri, M. R. N. Murthy, Bindu Pillai, null Kannan, M. V. Hosur, Mukesh Kumar, Swati Patwardhan, K. K. Kannan, B. Padmanabhaa, S. Sasaki-Sugio, M. Nukaga, T. Matsuzaki, S. Karthikevan, S. Sharma, A. K. Sharma, M. Paramasivam, P. Kumar, J. A. Khan, S. Yadav, A. Srinivasan, T. P. Singh, S. Gourinath, Neelima Alam, A. Srintvasan, Vikas Chandra, Punit Kaur, Ch. Betzel, S. Ghosh, A. K. Bera, S. Bhattacharya, S. Chakraborty, A. K. Pal, B. P. Mukhopadhyay, I. Dey, U. Haldar, Asok Baneriee, Jozef Sevcik, Adriana Solovicova, K. Sekar, M. Sundaralingam, N. Genov, Dong-cai Liang, Tao Jiang, Ji-ping Zhang, Wen-rui Chang, Wolfgang Jahnke, Marcel Blommers, S. C. Panchal, R. V. Hosur, Bindu Pillay, Puniti Mathur, S. Srivatsun, Ratan Mani Joshi, N. R. Jaganathan, V. S. Chauhan, H. S. Atreya, S. C. Sahu, K. V. R. Chary, Girjesh Govil, Elisabeth Adjadj, Éric Quinjou, Nadia Izadi-Pruneyre, Yves Blouquit, Joël Mispelter, Bernadette Heyd, Guilhem Lerat, Philippe Milnard, Michel Desmadreil, Y. Lin, B. D. Nageswara Rao, Vidva Raghunathan, Mei H. Chau, Prashant Pesais, Sudha Srivastava, Evans Coutinho, Anil Saran, Leizl F. Sapico, Jayson Gesme, Herbert Lijima, Raymond Paxton, Thamarapu Srikrishnan, C. R. Grace, G. Nagenagowda, A. M. Lynn, Sudha M. Cowsik, Sarata C. Sahu, S. Chauhan, A. Bhattacharya, G. Govil, Anil Kumar, Maurizio Pellecchia, Erik R. P. Zuiderweg, Keiichi Kawano, Tomoyasu Aizawa, Naoki Fujitani, Yoichi Hayakawa, Atsushi Ohnishi, Tadayasu Ohkubo, Yasuhiro Kumaki, Kunio Hikichi, Katsutoshi Nitta, V. Rani Parvathy, R. M. Kini, Takumi Koshiba, Yoshihiro Kobashigawa, Min Yao, Makoto Demura, Astushi Nakagawa, Isao Tanaka, Kunihiro Kuwajima, Jens Linge, Seán O. Donoghue, Michael Nilges, G. Chakshusmathi, Girish S. Ratnaparkhi, P. K. Madhu, R. Varadarajan, C. Tetreau, M. Tourbez, D. Lavalette, M. Manno, P. L. San Biagio, V. Martorana, A. Emanuele, S. M. Vaiana, D. Bulone, M. B. Palma-Vittorelli, M. U. Palma, V. D. Trivedi, S. F. Cheng, W. J. Chien, S. H. Yang, S. Francis, D. K. Chang, Renn Batra, Michael A. Geeves, Dietmar J. Manstein, Joanna Trvlska, Pawel Grochowski, Maciej Geller, K. Ginalski, P. Grochowski, B. Lesyng, P. Lavalette, Y. Blouquit, D. Roccatano, A. Amadei, A. Di Nola, H. J. C. Berendsen, Bosco Ho, P. M. G. Curmi, H. Berry, D. Lairez, E. Pauthe, J. Pelta, V. Kothekar, Shakti Sahi, M. Srinivasan, Anil K. Singh, Kartha S. Madhusudnan, Fateh S. Nandel, Harpreet Kaur, Balwinder Singh, D. V. S. Jain, K. Anton Feenstra, Herman J. C. Berendsen, F. Tama, Y. -H. Sanejouand, N. Go, Deepak Sharma, Sunita Sharma, Santosh Pasha, Samir K. Brahmachari, R. Viiavaraghavan, Jyoti Makker, Sharmisllia Dey, S. Kumar, G. S. Lakshmikanth, G. Krishnamoorthy, V. M. Mazhul, E. M. Zaitseva, Borys Kierdaszuk, J. Widengren, B. Terry, Ü. Mets, R. Rigler, R. Swaminathan, S. Thamotharan, N. Yathindra, Y. Shibata, H. Chosrowjan, N. Mataga, I. Morisima, Tania Chakraharty, Ming Xiao, Roger Cooke, Paul Selvin, C. Branca, A. Faraone, S. Magazù, G. Maisano, P. Migliardo, V. Villari, Digambar V. Behere, M. Sharique Zahida Waheed Deva, M. Brunori, F. Cutruzzolà, Q. H. Gibson, C. Savino, C. Travaglini-Allocatelli, B. Vallone, Swati Prasad, Shyamalava Mazumdar, Samaresh Mitra, P. Soto, R. Fayad, I. E. Sukovataya, N. A. Tyulkova, Sh. V. Mamedov, B. Aktas, M. Canturk, B. Aksakal, R. Yilgin, K. I. Bogutska, N. S. Miroshnichenko, S. Chacko, M. DiSanto, J. A. Hypolite, Y-M. Zheng, A. J. Wein, M. Wojciechowski, T. Grycuk, J. Antosiewicz, Marc A. Ceruso, Alfredo Di Nola, Subhasis Bandvopadhvay, Bishnu P. Chatterjee, Devapriva Choudhury, Andrew Thompson, Vivian Stojanoff, Jerome Pinkner, Scott Hultgren, Stefan Khight, Delphine Flatters, Julia Goodfellow, Fumi Takazawatt, Minoru Kanehisa, Masaki Sasai, Hironori Nakamura, Wang Bao Han, Yuan Zheng, Wang Zhi Xin, Pan xin Min, Vlnod Bhakuni, Sangeeta Kulkarni, Atta Ahmad, Koodathingal Prakash, Shashi Prajapati, Alexey Surin, Tomoharu Matsumoto, Li Yang, Yuki Nakagawa, Kazumoto Kimura, Yoshiyuki Amemiya, Gennady V. Semisotnov, Hiroshi Kihara, Saad Tayyab, Salman Muzammil, Yogesh Kumar, Vinod Bhakuni, Monica Sundd, Suman Kundu, M. V. Jagannadham, Medicherla V. Jagannadham, Bina Chandani, Ruby Dhar, Lalankumar Sinha, Deepti Warrier, Sonam Mehrotra, Purnima Khandelwal, Subhendu Seth, Y. U. Sasidhar, C. Ratna Prabha, Arun Gidwani, K. P. Madhusudan, Akira R. Kinjo, Ken Nishikawa, Suvobrata Chakravarty, Raghavan Varadarajan, K. Noyelle, P. Haezebrouck, M. Joniau, H. Van Dael, Sheffali Dash, Indra Brata Jha, Rajiv Bhat, Prasanna Mohanty, A. K. Bandyopadhyay, H. M. Sonawat, Ch. Mohan Rao, Siddhartha Datta, K. Rajaraman, B. Raman, T. Ramakrishna, A. Pande, J. Pande, S. Betts, N. Asherie, O. Ogun, J. King, G. Benedek, I. V. Sokolova, G. S. Kalacheva, Masashi Sonoyama, Yasunori Yokoyama, Kunihiro Taira, Shigeki Mitaku, Chicko Nakazawal, Takanori Sasakil, Yuri Mukai, Naoki Kamo, Seema Dalal, Lynne Regan, Shigeki Mituku, Mihir Roychoudhury, Devesh Kumar, Dénes Lőrinczv, Franciska Könczöl, László Farkas, Joseph Belagyi, Christoph Schick, Christy A. Thomson, Vettai S. Ananthanarayanan, E. G. Alirzayeva, S. N. Baba-Zade, M. Michael Gromiha, M. Oobatake, H. Kono, J. An, H. Uedaira, A. Sarai, Kazufumi Takano, Yuriko Yamagata, Katsuhide Yutani, Gouri S. Jas, Victor Muñoz, James Hofrichter, William A. Eaton, Jonathan Penoyar, Philip T. Lo Verde, J. Kardos, Á. Bódi, I. Venekei, P. Závodszky, L. Gráf, András Szilágyi, Péter Závodszky, R. D. Allan, J. Walshaw, D. N. Woolfson, Jun Funahashi, Savan Gupta, M. Mangoni, P. Roccatano, Gosu Ramachandraiah, Nagasuma R. Chandra, Barbara Ciani, Derek N. Woolfson, Usha B. Nair, Kanwal J. Kaur, Dinakar M. Salunke, Chittoor P. Swaminathan, Avadhesha Surolia, A. Pramanik, P. Jonasson, G. Kratz, O. T. Jansson, P. -Å. Nygren, S. Ståhl, K. Ekberg, B. -L. Johansson, S. Uhlén, M. Uhlén, H. Jörnvall, J. Wahren, Karin Welfle, Rolf Misselwitz, Wolfgang Höhne, Heinz Welfle, L. G. Mitskevich, N. V. Fedurkina, B. I. Kurganov, Gotam K. Jarori, Haripada Maity, J. Guharay, B. Sengupta, P. K. Sengupta, K. Sridevi, S. R. Kasturi, S. P. Gupta, Gunjan Agarwal, Suzanne Kwong, Robin W. Briehl, O. I. Ismailova, N, A. Tyulkova, C. Hariharan, D. Pines, E. Pines, M. Zamai, R. Cohen-Luria, A. Yayon, A. H. Parola, M. J. Padya, G. A. Spooner, D. N. Woolfeon, Panchan Bakshi, D. K. Bharadwaj, U. Sharma, N. Srivastava, R. Barthwal, N. R. Jagannathan, Keiko Matsuda, Takaaki Nishioka, Nobuhiro Go, T. Aita, S. Urata, Y. Husimi, Mainak Majumder, Nicola G. A. Abrescia, Lucy Malinina, Juan A. Subirana, Juan Aymami, Ramón Eritxa, Miquel Coll, B. J. Premraj, R. Thenmalarchelvi, P. Satheesh Kumar, N. Gautham, Lou -Sing Kan, null Ming-Hou, Shwu-Bin Lin, Tapas Sana, Kanal B. Roy, N. Bruant, D. Flatters, R. Lavery, D. Genest, Remo Rons, Heinz Sklenar, Richard Lavery, Sudip Kundu, Dhananjay Bhattacharyya, Debashree Bandyopadhyay, Ashoke Ranjan Thakur, Rabi Majumdar, F. Barceló, J. Portugal, Sunita Ramanathan, B. J. Rao, Mahua Gliosli, N. Vinay Kumar, Umesh Varshney, Shashank S. Pataskar, R. Sarojini, S. Selvasekarapandian, P. Kolandaivel, S. Sukumar, P. Kolmdaivel, Motilal Maiti, Anjana Sen, Suman Das, Elisa Del Terra, Chiara Suraci, Silvia Diviacco, Franco Quadrifoglio, Luigi Xodo, Arghya Ray, G. Karthikeyan, Kandala V. R. Chary, Basuthkar J. Rao, Anwer Mujeeb, Thomas L. James, N. Kasyanenko, E. E. F. Haya, A. Bogdanov, A. Zanina, M. R. Bugs, M. L. Cornélio, M. Ye. Tolstorukov, Nitish K. Sanval, S. N. Tiwari, Nitish K. Sanyal, Mihir Roy Choudhury, P. K. Patel, Neel S. Bhavesh, Anna Gabrielian, Stefan Wennmalm, Lars Edman, Rudolf Rigler, B. Constantinescu, L. Radu, I. Radulcscu, D. Gazdaru, Sebastian Wärmländer, Mikael Leijon, Setsuyuki Aoki, Takao Kondo, Masahiro Ishiura, V. A. Pashinskaya, M. V. Kosevich, V. S. Shelkovsky, Yu. P. Blagoy, Ji-hua Wang, R. Malathi, K. Chandrasekhar, E. R. Kandimalla, S. Agrawal, V. K. Rastogi, M. Alcolea Palafox, Chatar Singh, A. D. Beniaminov, S. A. Bondarenko, E. M. Zdobnov, E. E. Minyat, N. B. Ulyanov, V. I. Ivanov, J. S. Singh, Kailas D. Sonawane, Henri Grosjean, Ravindra Tewari, Uddhavesh B. Sonavane, Annie Morin, Elizabeth A. Doherty, Jennifer A. Doudna, H. Tochio, S. Sato, H. Matsuo, M. Shirakawa, Y. Kyogoku, B. Javaram, Surjit B. Dixit, Piyush Shukla, Parul Kalra, Achintya Das, Kevin McConnell, David L. Beveridge, W. H. Sawyer, R. Y. S. Chan, J. F. Eccelston, Yuling Yan, B. E. Davidson, Eimer Tuite, Bengt Norden, Peter Nielsen, Masayuki Takahashi, Anirban Ghosh, Manju Bansal, Frauke Christ, Hubert Thole, Wolfgang Wende, Alfred Pingoud, Vera Pingoud, Pratibha Mehta Luthra, Ramesh Chandra, Ranjan Sen, Rodney King, Robert Weisberg, Olaf F. A. Larsen, Jos Berends, Hans A. Heus, Cornelis W. Hilbers, Ivo H. M. van Stokkum, Bas Gobets, Rienk van Grondelle, Herbert van Amerongen, HE. Sngrvan, Yu. S. Babayan, N. V. Khudaverdian, M. Gromiha, F. Pichierri, M. Aida, P. Prabakaran, K. Sayano, Saulius Serva, Eglė Merkienė, Giedrius Vilkaitis, Elmar Weinhold, Saulius Klimašauskas, Eleonora Marsich, Antonella Bandiera, Giorgio Manzini, G. Potikyan, V. Arakelyan, Yu. Babayan, Alex Ninaber, Julia M. Goodfellow, Yoichiro Ito, Shigeru Ohta, Yuzuru Husimi, J. Usukura, H. Tagami, H. Aiba, Mougli Suarez, Elia Nunes, Deborah Keszenman, E. Carmen Candreva, Per Thyberg, Zeno Földes-Papp, Amita Joshi, Dinesh Singh, M. R. Rajeswari, null Ira, M. Pregetter, H. Amenitsch, J. Chapman, B. N. Pandev, K. P. Mishra, E. E. Pohl, J. Sun, I. I. Agapov, A. G. Tonevitsky, P. Pohl, S. M. Dennison, G. P. Gorbeako, T. S. Dynbko, N. Pappavee, A. K. Mishra, Prieto Manuel, Almeida Rodrigo, Loura Luis, L. Ya. Gendel, S. Przestalski, J. Kuczera, H. Kleszczyńska, T. Kral, E. A. Chernitsky, O. A. Senkovich, V. V. Rosin, Y. M. Allakhverdieva, G. C. Papageorgiou, R. A. Gasanov, Calin Apetrei, Tudor Savopol, Marius Balea, D. Cucu, D. Mihailescu, K. V. Ramanathan, Goran Bačić, Nicolas Sajot, Norbert Garnier, Serge Crouzy, Monique Genest, Z. S. Várkonyi, O. Zsiros, T. Farkas, Z. Combos, Sophie Cribier, I. F. Fraceto, S. Schreier, A. Spisni, F. de Paula, F. Sevšek, G. Gomišček, V. Arrigler, S. Svetina, B. Žekš, Fumimasa Nomura, Miki Nagata, Kingo Takiguchi, Hirokazu Hotani, Lata Panicker, P. S. Parvathanathan, A. Ishino, A. Saitoh, H. Hotani, K. Takiguchi, S. Afonin, A. Takahashi, Y. Nakato, T. Takizawa, Dipti Marathe, Kent Jørgensen, Satinder S. Rawat, R. Rukmini, Amitabha Chattopadhyay, M. Šentiurc, J. Štrancar, Z. Stolič, K. Filipin, S. Pečar, S. C. Biswas, Satyen Sana, Anunay Samanta, Koji Kinoshita, Masahito Yamazaki, Tetsuhiko Ohba, Tai Kiuchi, null Yoshitoshi, null Kamakura, Akira Goto, Takaaki Kumeta, Kazuo Ohki, I. P. Sugar, T. E. Thompson, K. K. Thompson, R. L. Biltonen, Y. Suezaki, H. Ichinose, M. Akivama, S. Matuoka, K. Tsuchihashi, S. Gasa, P. Mattjus, J. G. Molotkovsky, H. M. Pike, R. E. Brown, Ashish Arora, Jörg H. Kleinschmidt, Lukas K. Tamm, O. G. Luneva, K. E. Kruglyakova, V. A. Fedin, O. S. Kuptsoya, J. W. Borst, N. V. Visser, A. J. W. G. Visser, T. S. Dyubko, Toshihiko Ogihara, Kiyoshi Mishima, A. L. Shvaleva, N. Č. Radenović, P. M. Minić, M. G. Jeremić, Č. N. Radenović, T. F. Aripov, E. T. Tadjibaeva, O. N. Vagina, M. V. Zamaraeva, B. A. Salakhutdinov, A. Cole, M. Poppofl, C. Naylor, R. Titball, A. K. Basak, J. T. Eaton, C. E. Naylor, N. Justin, D. S. Moss, R. W. Titball, F. Nomura, M. Nagata, S. Ishjkawa, S. Takahashi, Kaoru Obuchi, Erich Staudegger, Manfred Kriechbaum, Robert I. Lehrer, Alan J. Waring, Karl Lohner, Susanne Gangl, Bernd Mayer, Gottfried Köhler, J. Shobini, Z. Guttenberg, B. Lortz, B. Hu, E. Sackmann, N. M. Kozlova, L. M. Lukyanenko, A. N. Antonovich, E. I. Slobozhanina, Andrey V. Krylov, Yuri N. Antonenko, Elena A. Kotova, Alexander A. Yaroslavov, Subhendu Ghosh, Amal K. Bera, Sudipto Das, Eva Urbánková, Masood Jelokhani-Niaraki, Karl Freeman, Petr Jezek, P. B. Usmanov, A. Ongarbaev, A. K. Tonkikh, Peter Pohl, Sapar M. Saparov, P. Harikumar, J. P. Reeves, S. Rao, S. K. Sikdar, A. S. Ghatpande, C. Corsso, A. C. Campos de Carvalho, W. A. Varanda, C. ElHamel, E. Dé, N. Saint, G. Molle, Anurae Varshney, M. K. Mathew, E. Loots, E. Y. Isacoff, Michiki Kasai, Naohiro Yamaguchi, Paramita Ghosh, Joseph Tigyi, Gabor Tigyi, Karoly Liliom, Ricardo Miledi, Maja R. Djurisic, Pavle R. Andjus, Indira H. Shrivastava, M. S. P. Sansom, C. Barrias, P. F. Oliveira, A. C. Mauricio, A. M. Rebelo da Costa, I. A. Lopes, S. V. Fedorovich, V. S. Chubanov, M. V. Sholukh, S. V. Konev, N. Fedirko, V. Manko, M. Klevets, N. Shvinka, B. S. Prabhananda, Mamata H. Kombrabail, S. Aravamudhan, Berenice Venegas-Cotero, Ivan Ortega Blake, Zhi-hong Zhang, Xiao-jian Hu, Han-qing Zhou, Wei-ying Cheng, Hang-fang Feng, L. O. Dubitsky, L. S. Vovkanvch, I. A. Zalyvsky, E. Savio-Galimberti, P. Bonazzola, J. E. Ponce-Homos, Mario Parisi, Claudia Capurro, Roxana Toriano, Laxma G. Ready, Larry R. Jones, David D. Thomas, B. A. Tashmukhamedov, B. T. Sagdullaev, D. Heitzmann, R. Warth, M. Bleich, R. Greger, K. T. G. Ferreira, H. G. Ferreira, Orna Zagoory, Essa Alfahel, Abraham H. Parola, Zvi Priel, H. Hama-Inaba, R. Wang, K. Choi, T. Nakajima, K. Haginoya, M. Mori, H. Ohyama, O. Yukawa, I. Hayata, Nanda B. Joshi, Sridhar K. Kannurpatti, Preeti G. Joshi, Mau Sinha, Xun Shen, Tianhui Hu, Ling Bei, Menno L. W. Knetsch, Nicole Schäfers, John Sandblom, Juris Galvanovskis, Roxana Pologea-Moraru, Eugenia Kovacs, Alexandra Dinu, S. H. Sanghvi, V. Jazbinšek, G. Thiel, W. Müller, G. Wübeller, Z. Tronteli, Leš Fajmut, Marko Marhl, Milan Brumen, I. D. Volotovski, S. G. Sokolovski, M. R. Knight, Alexei N. Vasil’ev, Alexander V. Chalyi, P. Sharma, P. J. Steinbach, M. Sharma, N. D. Amin, J. Barchir, R. W. Albers, H. C. Pant, M. Balasubramanyam, M. Condrescu, J. P. Gardner, Shamci Monajembashi, Gotz Pilarczyk, K. O. Greulich, F. M. El-Refaei, M. M. Talaat, A. I. El-Awadi, F. M. Ali, Ivan Tahradník, Jana Pavelková, Alexandra Zahradniková, Boris S. Zhorov, Vettai S. Ananthanaravanan, M. Ch. Michailov, E. Neu, W. Seidenbusch, E. Gornik, D. Martin, U. Welscher, D. G. Weiss, B. R. Pattnaik, A. Jellali, V. Forster, D. Hicks, J. Sahel, H. Dreyfus, S. Picaud, Hong-Wei Wang, Sen-fang Sui, Pradeep K. Luther, John Barry, Ed Morris, John Squire, C. Sivakama Sundari, D. Balasubramanian, K. Veluraia, T. Hema Thanka Christlet, M. Xavier Suresh, V. Laretta-Garde, Dubravka Krilov, Nataša Stojanović, Janko N. Herak, Ravi Jasuja, Maria Ivanova, Rossen Mirchev, Frank A. Ferrone, David Stopar, Ruud B. Spruijt, Cor J. A. M. Wolfs, Marcus A. Hemminga, G. Arcovito, M. De Spirito, Rajendra K. Agrawal, Amy B. Heagle, Pawel Penczek, Robert Grassucci, Joachim Frank, Manjuli R. Sharma, Loice H. Jeyakumar, Sidney Fleischer, Terence Wagenknecht, Carlo Knupp, Peter M. G. Munro, Eric Ezra, John M. Squire, Koji Ichihara, Hidefumi Kitazawa, Yusuke Iguchi, Tomohiko J. Itoh, Greta Pifat, Marina Kveder, Slavko Pečar, Milan Schara, Deepak Nair, Kavita Singh, Kanury V. S. Rao, Kanwaljeet Kaur, Deepti Jain, B. Sundaravadivel, Manisha Goel, D. M. Salunke, E. I. Kovalenko, G. N. Semenkova, S. N. Cherenkevich, T. Lakshmanan, D. Sriram, S. Srinivasan, D. Loganathan, T. S. Ramalingam, J. A. Lebrón, P. J. Bjorkman, A. K. Singh, T. N. Gayatri, Ernesto R. Caffarena, J. Raul Grigera, Paulo M. Bisch, V. Kiessling, P. Fromherz, K. N. Rao, S. M. Gaikwad, M. I. Khan, C. G. Suresh, P. Kaliannan, M. Elanthiraiyan, K. Chadha, J. Payne, J. L. Ambrus, M. P. N. Nair, Madhavan P. N. Nair, S. Mahajan, K. C. Chadha, R. Hewitt, S. A. Schwartz, J. Bourguignon, M. Faure, C. Cohen-Addad, M. Neuburger, R. Ober, L. Sieker, D. Macherel, R. Douce, D. S. Gurumurthy, S. Velmurugan, Z. Lobo, Ratna S. Phadke, Prashant Desai, I. M. Guseinova, S. Yu. Suleimanov, I. S. Zulfugarov, S. N. Novruzova, J. A. Aliev, M. A. Ismayilov, T. V. Savchenko, D. R. Alieva, Petr Ilík, Roman Kouřil, Hana Bartošková, Jan Nauš, Jvoti U. Gaikwad, Sarah Thomas, P. B. Vidyasagar, G. Garab, I. Simidjiev, S. Rajagopal, Zs. Várkonyi, S. Stoylova, Z. Cseh, E. Papp, L. Mustárdy, A. Holzenburg, R. Bruder, U. K. Genick, T. T. Woo, D. P. Millar, K. Gerwert, E. D. Getzoff, Tamás Jávorfí, Győző Garab, K. Razi Naqvi, Md. Kalimullah, Jyoti Gaikwad, Manoj Semwal, Roman Kouril, Petr Ilik, Man Naus, István Pomozi, Gábor Horváth, Rüdiger Wehner, Gary D. Bernard, Ana Damjanović, Thorsten Ritz, Klaus Schulten, Wang Jushuo, Shan Jixiu, Gong Yandao, Kuang Tingyun, Zhao Nanming, Arvi Freiberg, Kõu Timpmann, Rein Ruus, Neal W. Woodbury, E. V. Nemtseva, N. S. Kudryasheva, A. G. Sizykh, V. N. Shikhov, T. V. Nesterenko, A. A. Tikhomirov, Giorgio Forti, Giovanni Finazzi, Alberto Furia, Romina Paola Barbagallo, S. Iskenderova, R. Agalarov, R. Gasanov, Miyashita Osamu, G. O. Nobuhiro, R. K. Soni, M. Ramrakhiani, Hiromasa Yagi, Kacko Tozawa, Nobuaki Sekino, Tomoyuki Iwabuchi, Masasuke Yoshida, Hideo Akutsu, A. V. Avetisyan, A. D. Kaulen, V. P. Skulachev, B. A. Feniouk, Cécile Breyton, Werner Kühlbrandt, Maria Assarsson, Astrid Gräslund, G. Horváth, B. Libisch, Z. Gombos, N. V. Budagovskaya, N. Kudryasheva, Erisa Harada, Yuki Fukuoka, Tomoaki Ohmura, Arima Fukunishi, Gota Kawai, Kimitsuna Watanabe, Jure Derganc, Bojan Božič, Saša Svetina, Boštjan Žekš, J. F. Y. Hoh, Z. B. Li, G. H. Rossmanith, E. L. de Beer, B. W. Treijtel, P. L. T. M. Frederix, T. Blangè, S. Hénon, F. Galtet, V. Laurent, E. Planus, D. Isabey, L. S. Rath, P. K. Dash, M. K. Raval, C. Ramakrishnan, R. Balaram, Milan Randic, Subhash C. Basak, Marjan Vracko, Ashesh Nandy, Dragan Amic, Drago Beslo, Sonja Nikolic, Nenad Trinajstic, J. Walahaw, Marc F. J. Lensink, Boojala V. B. Reddy, Ilya N. Shindylov, Philip E. Bourne, M. C. Donnamaria, J. de Xammar Oro, J. R. Grigera, Monica Neagu, Adrian Neagu, Matej Praprotnik, Dušanka Janežič, Pekka Mark, Lennart Nilsson, L. La Fata, Laurent E. Dardenne, Araken S. Werneck, Marçal de O. Neto, N. Kannan, S. Vishveshwara, K. Veluraja, Gregory D. Grunwald, Alexandra T. Balaban, Kanika Basak, Brian D. Gute, Denise Mills, David Opitz, Krishnan Balasubramanian, G. I. Mihalas, Diana Lungeanu, G. Macovievici, Raluca Gruia, C. Cortez-Maghelly, B. Dalcin, E. P. Passos, S. Blesic, M. Ljubisavljevic, S. Milosevic, D. J. Stratimirovic, Nandita Bachhawat, Shekhar C. Mande, A. Nandy, Ayumu Saito, Koichi Nishigaki, Mohammed Naimuddin, Takatsugu Hirokawa, Mitsuo Ono, Hirotomo Takaesu, M. I. El Gohary, Abdalla S. Ahmed, A. M. Eissa, Hiroshi Nakashima, G. P. S. Raghava, N. Kurgalvuk, O. Goryn, Bernard S. Gerstman, E. V. Gritsenko, N. N. Remmel, O. M. Maznyak, V. A. Kratasyuk, E. N. Esimbekova, D. Tchitchkan, S. Koulchitsky, A. Tikhonov, A. German, Y. Pesotskaya, S. Pashkevich, S. Pletnev, V. Kulchitsky, Umamaheswar Duvvuri, Sridhar Charagundla, Rahim Rizi, John S. Leigh, Ravinder Reddy, Mahesh Kumar, O. Coshic, P. K. Julka, O. K. Rath, NR. Jagannathan, Karina Roxana Iliescu, Maria Sajin, Nicolcta Moisoi, Ileana Petcu, A. I. Kuzmenko, R. P. Morozova, I. A. Nikolenko, G. V. Donchenko, M. K. Rahman, M. M. Ahmed, Takehiro Watanabe, Y. Rubin, H. Gilboa, R. Sharony, R. Ammar, G. Uretzky, M. Khubchandani, H. N. Mallick, V. Mohan Kumar, Arijitt Borthakur, Erik M. Shapiro, M. Gulnaz Begum, Mahaveer N. Degaonkar, S. Govindasamy, Ivan Dimitrov, T. A. Kumosani, W. Bild, I. Stefanescu, G. Titescu, R. Iliescu, C. Lupusoru, V. Nastasa, I. Haulica, Gopal Khetawat, N. Faraday, M. Nealen, S. Noga, P. F. Bray, T. V. Ananieva, E. A. Lycholat, MV. Kosevich, S. G. Stepanyan, S. V. Antonyuk, R. Khachatryan, H. Arakelian, A. Kumar, S. Ayrapetyan, V. Mkheyan, S. Agadjanyan, A. Khachatryan, S. S. Rajan, V. Kabaleeswaran, Geetha Gopalakrishnan, T. R. Govindachari, Meera Ramrakhiani, Phillip Lowe, Andrew Badley, David C. Cullen, H. Hermel, W. Schmahl, H. Möhwald, Nirmalya Majumdar, Joydip Das, András Dér, Loránd Kelemen, László Oroszi, András Hámori, Jeremy J. Ramsden, Pál Ormos, D. Savitri, Chanchal K. Mitra, Toshio Yanagida, Seiji Esaki, Yuji Kimura, Tomoyuki Nishida, Yosiyuki Sowa, M. Radu, V. K. Koltover, Ya. I. Estrin, L. A. Kasumova, V. P. Bubnov, E. E. Laukhina, Rajiv Dotta, M. Degaonkar, P. Raghunathan, Rama Jayasundar, Pavel Novák, Milan Marko, Ivan Zahradník, Hiroaki Hirata, Hidetake Miyata, J. Balaji, P. Sengupta, S. Maiti, M. Gonsalves, A. L. Barker, J. V. Macpherson, D. O’Hare, C. P. Winlove, P. R. Unwin, R. Phillip, S. Banerjee, G. Ravindra Kumar, K. Nagayaka, R. Danev, S. Sugitani, K. Murata, Michael Gősch, H. Blom, P. Thyberg, Z. Földes-Papp, G. Björk, J. Holm, T. Heino, Masashi Yokochi, Fuyuhiko Inagaki, Masami Kusunoki, E. K. Matthews, J. Pines, Yu. P. Chukova, Vitaly K. Koltover, Geetanjali Bansal, Uma Singh, M. P. Bansal, Kotoko Nakata, Tastuya Nakano, Tsuguchika Kaminuma, B. P. S. Kang, U. Singh, Bonn Kirn, Neja Potocnik, Vito Stare, Latal Shukla, V. Natarajan, T. P. A. Devasagayam, M. D. Sastry, P. C. Kesavan, R. Sayfutdinov, V. V. Adamovich, D. Yu. Rogozin, A. G. Degermendzhy, C. L. Khetrapal, G. A. Nagana Gowda, Kedar Nath Ghimire, Ishida Masaru, H. Fujita, S. Ishiwata, Y. Kishimoto, S. Kawahara, M. Suzuki, H. Mori, M. Mishina, Y. Kirino, H. Ohshima, A. S. Dukhin, V. N. Shilov, P. J. Goetz, and R. K. Mishra

Subjects
0303 health sciences, biology, General Medicine, 010402 general chemistry, 01 natural sciences, Horseradish peroxidase, General Biochemistry, Genetics and Molecular Biology, 0104 chemical sciences, 03 medical and health sciences, Biochemistry, Manganese porphyrin, biology.protein, Enzyme reconstitution, General Agricultural and Biological Sciences, 030304 developmental biology
Published
1999
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8. How to Evaluate the Contribution of Factors to the Conformational Stability of a Protein

Author

Jun Funahashi, Katsuhide Yutani, and Kazufumi Takano

Subjects
Hydrophobic effect, Crystallography, Chemistry, Hydrogen bond, Mutant, Biophysics, A protein, Molecule, Denaturation (biochemistry), Conformational stability, Crystal structure
Abstract

Mutant proteins are useful for study of protein stability. However, it is difficult to explain the changes in stability due to mountain, because contributions of amino acid residues to the conformational stability are quite different, depending on their location. In order to quantify directly the contribution of several factors to the conformational stability of a protein, we determined the thermodynamic parameters of denaturation and the crystal structures for more than 60 mutant human lysozymes. Analyzing this database (stability/structure database), the contribution of some factors to conformational stability of a protein, such as hydrophobic effect, hydrogen bond, and water molecule, could be estimated.

Published
1999
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9. Contribution of Hydrogen Bonds to the Conformational Stability of Human Lysozyme: Calorimetry and X-ray Analysis of Six Tyrosine → Phenylalanine Mutants

Author

Yohei Sumikawa, Kazufumi Takano, Yuriko Yamagata, Katsuhide Yutani, Masahiro Kubota, Jun Funahashi, and Satoshi Fujii

Subjects
Models, Molecular, Protein Denaturation, Protein Folding, Calorimetry, Differential Scanning, Protein Conformation, Hydrogen bond, Stereochemistry, Chemistry, Phenylalanine, Hydrogen Bonding, Crystallography, X-Ray, Biochemistry, Crystallography, chemistry.chemical_compound, Protein structure, Intramolecular force, Hydrolase, Mutagenesis, Site-Directed, Humans, Tyrosine, Molecule, Muramidase, Protein folding, Lysozyme
Abstract

The contribution of hydrogen bonds to the conformational stability of human lysozyme was investigated by the combination of calorimetric and X-ray analyses of six Tyr --> Phe mutants. Unfolding Delta G and unfolding Delta H values of the Tyr --> Phe mutant proteins were changed by from +0.3 to -4.0 kJ/mol and from 0 to -16 kJ/mol, respectively, compared to those of the wild-type protein. The net contribution of a hydrogen bond at a specific site to stability (Delta Gwild/HB), considering factors affected by substitutions, was evaluated on the basis of X-ray structures of the mutant proteins. In the present study, one of six mutant proteins was suitable for evaluating the strength of the hydrogen bond. Delta Gwild/HB for the intramolecular hydrogen bond at Tyr124 was evaluated to be 7.5 kJ/mol. Results of the analysis of other mutants also suggest that hydrogen bonds of the hydroxyl group of Tyr, including the hydrogen bond with a water molecule, contribute to the stabilization of the human lysozyme.

Published
1998
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10. Contribution of water molecules in the interior of a protein to the conformational stability

Author

Jun Funahashi, Yuriko Yamagata, Katsuhide Yutani, Satoshi Fujii, and Kazufumi Takano

Subjects
chemistry.chemical_classification, Calorimetry, Differential Scanning, Protein Conformation, Globular protein, Mutant, Water, Crystal structure, Crystallography, X-Ray, Crystallography, Amino Acid Substitution, Drug Stability, chemistry, Structural Biology, Mole, Hydrolase, Mutagenesis, Site-Directed, Humans, Thermodynamics, Molecule, Muramidase, Denaturation (biochemistry), Conformational stability, Molecular Biology
Abstract

Water molecules frequently occur in the interior of globular proteins. To elucidate the contribution of buried water molecules to the conformational stability of a protein, we examined the crystal structures and the thermodynamic parameters of denaturation of six Ile to Ala/Gly mutant human lysozymes, in which a cavity is created at each mutation site by the substitution of a smaller side-chain for a larger one. One or two ordered water molecules were found in the cavities created in some mutants (I106A, I59A and I59G). The cavity volumes for these three mutants were bigger than those that remained empty in the other mutants. The stability of the mutant proteins with the newly introduced water molecules was about 8 kJ/mol higher than that expected from the change in hydrophobic surface area (DeltaDeltaASAHP) exposed upon denaturation. It was concluded that a water molecule in a cavity created in the interior of a protein contributes favorably to the stability.

Published
1997
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11. Mechanism underlying the block of human Cav3.2 T-type Ca2+ channels by benidipine, a dihydropyridine Ca2+ channel blocker

Author

Yoshiyuki Sugimoto, Jun Funahashi, Saori Takahashi, Masahiro Matsubara, Hideaki Kusaka, and Atsushi Inayoshi

Subjects
Dihydropyridines, HEK 293 cells, Dihydropyridine, Efonidipine, Depolarization, Stimulation, General Medicine, Calcium Channel Blockers, General Biochemistry, Genetics and Molecular Biology, chemistry.chemical_compound, Calcium Channels, T-Type, HEK293 Cells, chemistry, Benidipine, medicine, Biophysics, Humans, Ca2 channels, Channel blocker, General Pharmacology, Toxicology and Pharmaceutics, Ion Channel Gating, medicine.drug
Abstract

Aims Benidipine, a dihydropyridine Ca 2+ channel blocker, has been reported to block T-type Ca 2+ channels; however, the mechanism underlying this effect was unclear. In this study, we characterized the mechanism responsible for this blocking activity. Furthermore, the blocking activity was compared between two enantiomers of benidipine, (S, S)- and (R, R)-benidipine. Main methods Human Ca v 3.2 (hCa v 3.2) T-type Ca 2+ channels stably expressed in the human embryonic kidney cell line, HEK-293, were studied in whole-cell patch-clamp recordings and Ca 2+ mobilization assay. Key findings In whole-cell patch-clamp recordings, benidipine blocked hCa v 3.2 T-type Ca 2+ currents elicited by depolarization to a comparable extent as efonidipine. The block was dependent on stimulation frequency and holding potential, but not test potential. Benidipine significantly shifted the steady-state inactivation curve to the hyperpolarizing direction, but had no effect on the activation curve. Benidipine prolonged the recovery from inactivation of hCa v 3.2 T-type Ca 2+ channels without any effect on the kinetics of activation, inactivation, or deactivation. In the Ca 2+ mobilization assay, benidipine was more potent than efonidipine in blocking Ca 2+ influx through hCa v 3.2 T-type Ca 2+ channels. (S, S)-Benidipine was more potent than (R, R)-benidipine in blocking hCa v 3.2 T-type Ca 2+ currents, but there was no difference in blocking the Ca 2+ influx. Significance We have characterized the blocking activity of benidipine against hCa v 3.2 Ca 2+ channels and revealed the difference between the two enantiomers of benidipine. The blocking action of benidipine could be mediated by stabilizing hCa v 3.2 Ca 2+ channels in an inactivated state.

Published
2010

12. Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity

Author

Jun Funahashi, Makoto Suzuki, Yoshinori Yamashita, Yumiko Uochi, Masami Kaneko, Michio Ichimura, Shiro Soga, Hideyuki Onodera, Yuichi Takahashi, Endang Sutriswati Rahayu, Shun-Ichi Ikeda, Takayuki Nakashima, and Yutaka Kanda

Subjects
Models, Molecular, Molecular model, Stereochemistry, Lactams, Macrocyclic, Clinical Biochemistry, Pharmaceutical Science, Antineoplastic Agents, Biochemistry, Streptomyces, chemistry.chemical_compound, Cell Line, Tumor, Drug Discovery, polycyclic compounds, Benzoquinones, Humans, HSP90 Heat-Shock Proteins, Cytotoxicity, Molecular Biology, biology, Molecular Structure, Chemistry, Ansamycin, Organic Chemistry, Geldanamycin, biology.organism_classification, Benzoquinone, Hsp90, Chaperone (protein), biology.protein, Molecular Medicine
Abstract

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1–A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1–A4 also adds to their potential as lead compounds for anti-tumor drugs.

Published
2007

13. The noncompetitive antagonism of histamine H1 receptors expressed in Chinese hamster ovary cells by olopatadine hydrochloride: its potency and molecular mechanism

Author

Yuichi, Matsumoto, Jun, Funahashi, Kiyotoshi, Mori, Kozue, Hayashi, and Hiroshi, Yano

Subjects
Umbilical Veins, Dose-Response Relationship, Drug, Endothelial Cells, CHO Cells, Binding, Competitive, Cricetulus, Isomerism, Cricetinae, Histamine H1 Antagonists, Animals, Humans, Calcium, Receptors, Histamine H1, Olopatadine Hydrochloride, Cells, Cultured, Dibenzoxepins, Histamine, Protein Binding
Abstract

Calcium responses to various concentrations of histamine were monitored in Chinese hamster ovary cells stably expressing the human histamine H(1) receptor. The effects of various histamine H(1) receptor antagonists on the dose-response curve for histamine were evaluated. Olopatadine hydrochloride (olopatadine) inhibited the histamine-induced maximum response (pD(2)': 7.5) but had insignificant effects on histamine EC(50) values. This noncompetitive property exhibited by olopatadine, which was also observed in human umbilical vein endothelial cells, was the most striking among the antihistamines tested in this study. The geometrical isomer of olopatadine (E-isomer), which had a similar binding affinity to the histamine H(1) receptor as olopatadine, showed a mixed antagonistic profile (competitive and noncompetitive). These results indicate that the geometry around the double bond in the dimethylaminopropylidene group is critical for the potent noncompetitive property of olopatadine. Furthermore, binding mode analyses suggest that the protonated amine group in the dimethylaminopropylidene moiety of olopatadine forms an ionic bond with Glu 181 that is present in the second extracellular loop of the histamine H(1) receptor, whereas the amine group of the E-isomer does not. The second extracellular loop in aminergic G-protein-coupled receptors contributes to ligand binding and therefore the noncompetitive property of olopatadine may be explained by the interaction with Glu 181.

Published
2007

14. Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Author

Jun Funahashi, Yuriko Yamagata, Katsuhide Yutani, and Kazufumi Takano

Subjects
Models, Molecular, Stereochemistry, Protein Conformation, Calorimetry, Crystal structure, Arginine, Crystallography, X-Ray, Biochemistry, chemistry.chemical_compound, Protein structure, Hydrolase, Serine, Molecule, Humans, Denaturation (biochemistry), Molecular Biology, Aspartic Acid, Chemistry, Hydrogen bond, Water, Valine, Cell Biology, Crystallography, Mutation, Thermodynamics, Tyrosine, Muramidase, Lysozyme, Asparagine, Protein Binding
Abstract

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.

Published
2002

15. Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,)

Author

Jun Funahashi, Yuriko Yamagata, Katsuhide Yutani, Yusaku Hioki, Kazufumi Takano, and Seiki Kuramitsu

Subjects
chemistry.chemical_classification, Models, Molecular, Calorimetry, Differential Scanning, Hydrogen bond, Chemistry, Stereochemistry, Globular protein, Protein Conformation, Intermolecular force, Hydrogen Bonding, Conformational entropy, Crystallography, X-Ray, Biochemistry, Recombinant Proteins, Crystallography, Protein structure, Amino Acid Substitution, Intramolecular force, Enzyme Stability, Molecule, Humans, Denaturation (biochemistry), Muramidase
Abstract

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

Published
1999

21. The structure, stability, and folding process of amyloidogenic mutant human lysozyme

Author

Katsuhide Yutani, Kazufumi Takano, Kyoko Ogasahara, Yoriko Yamagata, and Jun Funahashi

Subjects
Models, Molecular, Protein Denaturation, Amyloid, Protein Conformation, Hydrogen bond, Mutant, Amyloidosis, General Medicine, Crystallography, X-Ray, Biochemistry, Folding (chemistry), chemistry.chemical_compound, Residue (chemistry), chemistry, Mutagenesis, Mutant protein, Native state, Biophysics, Humans, Thermodynamics, Muramidase, Lysozyme, Molecular Biology
Abstract

The physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.

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