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2. Proposal of a Laboratory Course Dedicated to the Generation of Protein Molecular Weight Standards for Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Author

Bubis, José

Abstract

Protein molecular weight standards are routinely employed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the apparent sizes of unknown proteins within a sample. For training students, a laboratory course based on the production of marker proteins is proposed. Following fractionation by column chromatography, a series of purified proteins and mixtures of proteins were combined to generate various sets of unstained, prestained and fluorescently labeled molecular weight ladders for SDS-PAGE. The produced material can be used in successive laboratory practices as an alternative to commercial protein markers.

Published
2021
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4. The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides

Author

Bubis, José, Martínez, Juan Carlos, Calabokis, Maritza, Ferreira, Joilyneth, Sanz-Rodríguez, Carlos E, Navas, Victoria, Escalona, José Leonardo, Guo, Yurong, and Taylor, Susan S

Subjects
Biochemistry and Cell Biology, Biological Sciences, Rare Diseases, Amino Acid Sequence, Base Sequence, Cyclic AMP-Dependent Protein Kinases, Molecular Docking Simulation, Protein Binding, Protein Conformation, Sequence Homology, Nucleic Acid, Trypanosoma, Trypanosoma equiperdum, Regulatory subunits of the cAMP-dependent protein kinase, Gene cloning, Protein expression in bacteria, Protein purification, Biochemical characterization, Dourine, Trypanosomatid parasites, Biochemistry & Molecular Biology, Biochemistry and cell biology
Abstract

The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)8], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)8 was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)8 did not bind either [3H] cAMP or [3H] cGMP up to concentrations of 0.40 and 0.65 μM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA RIα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.

Published
2018

36. MPSA short communications

Author

Barnes, Jr., Eugene M., Calkin, Patricia A., Kuroda, Satoshi, Norioka, Shigemi, Mitta, Masanori, Kato, Ikunoshin, Sakiyama, Fumio, Nika, Heinz, Chow, David T., Hess, Daniel, Bures, Edward J., Morrison, Hamish D., Aebersold, Ruedi, Clore, G. Marius, Gronenborn, Angela M., Persson, Bengt, Argos, Patrick, James, Peter, Cannons, Andrew C., Solomonson, Larry P., Dombrowski, Kenneth E., Moddeman, William E., Wright, Stephen E., Barker, Winona C., George, David G., Mattagajasingh, Subhendra N., Misra, Hara P., Huang, Shuan Shian, Huang, Jung San, Lee, Y. C., Fischer, Wolfgang H., Craig, A. Grey, McFadden, Philip N., Lind-quist, Jonathan A., Bartlet-Jones, M., Jeffery, W., Hansen, H. F., Pappin, D. J. C., Bergman, Tomas, Hjelmqvist, Lars, Estonius, Mats, Jörnvall, Hans, Dorow, Donna S., Tschesche, H., Knäuper, V., Kleine, T., Reinemer, P., Schnierer, S., Grams, F., Bode, W., Southan, Christopher, Fantom, Kenneth, Lavery, Patric, Findlay, J. B. C., Akrigg, D., Attwood, T. K., Beck, M. J., Bleasby, A. J., North, A. C. T., Parry-Smith, D. J., Perkins, D. N., Aitken, A., Patel, Y., Martin, H., Jones, D., Robinson, K., Madrazo, J., Howell, S., Aebersold, Ruedi, Hess, Daniel, Yungwirth, Tom, Affolter, Michael, Amankwa, Lawrence, Hess, Daniel, Aebersold, Ruedi, Scheraga, Harold A., Yang, Chao -Yuh, Valentinova, Natalia V., Yang, Manlan, Gu, Zi -Wei, Gotto, Jr., Antonio M., Dovichi, Norman J., Waldron, Karen C., Chen, Min, Ireland, Ian, Omori, Akira, Yoshida, Sachiyo, Schaller, Johann, Lengweiler, Stephan, Rickli, Egon E., Bubis, José, Ortiz, Julio O., Möller, Carolina, Millán, Enrique J., Boyd, Victoria L., Bozzini, MeriLisa, Zhao, Jindong, DeFranco, Robert J., Yuan, Pau -Miau, Loudon, G. Marc, Nguyen, Duy, Kamo, Masaharu, Kawakami, Takao, Miyatake, Norifumi, Tsugita, Akira, Tsugita, Akira, Kamo, Masaharu, Takamoto, Keiji, Satake, Kazuo, Bischof, Oliver, Hechenberger, Mirko, Thiede, Bernd, Kruft, Volker, Wittmann-Liebold, Brigitte, Otto, Albrecht, Benndorf, Rainer, Wittmann-Liebold, Brigitte, Jungblut, Peter, Ühlein, Monika, Wittmann-Liebold, Brigitte, Urlaub, Henning, Kruft, Volker, Wittmann-Liebold, Brigitte, Berhardt, Rita, Kraft, Regine, Uhlmann, Heike, Beckert, Vita, Akizawa, Toshifumi, Ayabe, Takaaki, Matsukawa, Motomi, Itoh, Michiyasu, Nishi, Masatoshi, Sato, Hiroshi, Seiki, Motoharu, Yoshioka, Masanori, Lebl, Michal, Krchňák, Viktor, Sepetov, Nikolai F., Kočiš, Petr, Pátek, Marcel, Flegelová, Zuzana, Ferguson, Ronald, Lam, Kit S., Moritz, Robert L., Eddes, James, Ji, Hong, Reid, Gavin E., Simpson, Richard J., Seffens, William, Poulter, C. Dale, Dolence, Julia M., Bond, Pamela D., Nokihara, Kiyoshi, Ikegaya, Kazuo, Morita, Naoki, Ohmura, Takao, Salikhov, S. I., Sagdiev, N. J., Korneev, A. S., Dolimbek, Behzod Z., Atassi, M. Zhouhair, Rosenberg, J. S., Yun, Z., Wyde, P. R., Atassi, M. Z., Gaskell, Simon J., Anumula, Kalyan Rao, Goldenberg, David P., Appella, Ettore, Fiscella, Michelle, Zambrano, Nicola, Ullrich, Stephen J., Sakaguchi, Kazuyasu, Sakamoto, Hiroshi, Lewis, Marc S., Lin, David, Mercer, W. Edward, Anderson, Carl W., Anderson, Carl W., Connelly, Marjorie A., Zhang, Hong, Sipley, John D., Lees-Miller, Susan P., Sakaguchi, Kazuyasu, Ullrich, Stephen J., Jackson, Stephen P., Appella, Ettore, Xie, Yong-hong, Yang, Manlan, Quion, Jun A., Gotto, Jr., Antonio M., Yang, Chao-yuh, Brandt, W. F., Alk, H., Bhaskaran, R., Yu, Chin, Yang, C. C., Henschen, Agnes H., Ashman, Keith, Mann, Matthias, Guevara, Jr., Juan, Nguyen, Hung Michael, Davison, Daniel B., Morrisett, Joel D., Perham, Richard N., Marvin, Donald A., Symmons, Martyn F., Terry, Tamsin D., Beg, Z. H., Stonik, J. A., Hoeg, J. M., Brewer, Jr., H. B., Gorovits, Boris M., Raman, C. S., and Horowtiz, Paul M.

Published
1994
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45. Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate affects its functionality.

Author

Medina, Rafael, Perdomo, Deisy, Möller, Carolina, and Bubis, José

Subjects
RHODOPSIN, PHOTORECEPTORS, POLYACRYLAMIDE gel electrophoresis, ABSORPTION spectra
Abstract

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ~2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-crosslinked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl ß-D-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248. [ABSTRACT FROM AUTHOR]

Published
2020
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50. The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides

Author

Bubis, José, primary, Martínez, Juan Carlos, additional, Calabokis, Maritza, additional, Ferreira, Joilyneth, additional, Sanz-Rodríguez, Carlos E., additional, Navas, Victoria, additional, Escalona, José Leonardo, additional, Guo, Yurong, additional, and Taylor, Susan S., additional

Published
2018
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